IMMUNOLOGYORIGINAL ARTICLE

Tumour necrosis factor-a-mediated human polymeric immunoglobulin receptor expression is regulated by both mitogen-activatedprotein kinase and phosphatidylinositol-3-kinase in HT-29 cell line
N. Takenouchi-Ohkubo,1 I. Moro,2S. Mukae,1 Y. Kaneko1 andK. Komiyama1
1

Department of Pathology, Nihon University,
School of Dentistry, Tokyo, Japan and2
Advanced Research Institute for the Sciencesand Humanities, Nihon University, Tokyo,Japan

doi:10.1111/j.1365-2567.2007.02716.x
Received 5 March 2007; revised 25 July 2007;
accepted 14 August 2007.
Correspondence: Dr N. Takenouchi-Ohkubo,
Department of Pathology, Nihon University,
School of Dentistry, 1-8-13, KandaSurugadai, Chiyoda-ku, Tokyo 101–8310,
Japan. Email: takenouchi@dent.nihon-u.ac.jp
Senior author: Nobuko Takenouchi-Ohkubo

Summary
Human polymeric immunoglobulin receptor (pIgR) is present on the surface of glandular epithelium, and it plays a crucial role in the mucosal
immune defence. pIgR expression in HT-29 cells is up-regulated by oneof the proinflammatory cytokines, tumour necrosis factor (TNF)-a. However, the mechanism used by the TNF-a-mediated signalling pathway has
not been examined exclusively. To elucidate this mechanism in detail,HT-29 cells were cotreated with TNF-a and mitogen-activated protein
kinase kinase (MAPKK, also called MEK1) inhibitor, PD98059, and theamount of free secretory component (SC) secreted into the culture medium was measured. The amount of free SC stimulated by TNF-a wasincreased by addition of PD98059. This up-regulation occurred at thetranscriptional level. The amount of SC was also up-regulated by additionof TNF-a with U0126, an inhibitor of MEK1 and MEK2. Nuclear factor(NF)-jB activity and NF-jB binding to the jB2 site localized upstream ofthe pIgR gene did not change after coincubation of HT-29 cells with
TNF-a and PD98059. The expression level of pIgR by TNF-a wasdecreased by LY294002, an inhibitor of phosphatidylinositol-3-kinase(PI3K), at the transcriptional level. Extracellular signal-regulated kinase(ERK)1/2 phosphorylation and NF-jB binding to the jB2 site were notaffected by LY294002 treatment. These data suggest that TNF-a-mediated
pIgR expression is negatively regulated by ERK pathway, which is independent of NF-jB. In addition, decrease of SC production by Ly294002
suggests that the presence of PI3K mediated regulation of SC production.
Keywords: pIgR; TNF-a; MAPK; PI3K; NF-jB

Introduction
Tumour necrosis factor-a (TNF-a) has pleiotropic functions that include roles in cell growth, differentiation,
inflammatory effects, tumorigenesis, and viral replication.1
TNF-a has been identified as the most important cytokine
in the pathogenesis of inflammatory bowel disease(IBD).2 The effect of TNF-a is mediated through two
types of receptors, TNF-receptor (TNFR) 1 (also known
as p55TNFR) and TNFR2 (also known as p75TNFR).

TNF-a binding to these receptors activates transcription
factors such as nuclear factor (NF)-jB or AP-1 that control multiple gene expressions of inflammatory cytokines
such as interleukin (IL)-1, IL-6, and IL-8. TNF-a signals
mediated through the TNFRs are followed by activationof mitogen-activated protein kinase (MAPK) pathways,
including extracellular signal-regulated kinases (ERKs),
p38, and c-Jun NH2-terminal kinases.3
Human polymeric immunoglobulin receptor (pIgR) is a
120 000 MW glycoprotein present on glandular epithelial

Abbreviations: ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; ERK, extracellularsignal-regulated kinase; IBD, inflammatory bowel disease; IL-1b, interleukin-1 b; IL-6, interleukin-6; IL-8, interleukin-8; IFN-c,interferon-c; IRF-1, IFN-c regulatory factor-1; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; NF-jB,
nuclear factor-jB; PI3K, phosphatidylinositol-3-kinase; pIgR, polymeric immunoglobulin receptor; SC, secretory component;
S-IgA, secretory immunoglobulin A; TNF-a, tumor necrosis factor-a; TNFR, TNF-receptor.
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 2007 Blackwell Publishing Ltd, Immunology, 123, 500–507

pIgR regulation by MAPK and PI3K
cells that functions as a receptor for polymeric immunoglobulin (pIg). pIgR transports polymeric immunoglobulin A (IgA) into external secretions as secretory IgA(S-IgA), which is critical for the defence of mucosal tissues.4 Free secretory component (SC) is important for theenhancement of immune responses. For example, degranulation of eosinophils caused by binding of S-IgA or
SC is mediated through the 15 000 MW SC receptor
expressed in eosinophils.5,6 Degranulation of IL-3-primed
basophils is also mediated by SC.7 Free SC can bind toseveral bacterial proteins, such as colonization factor antigen,8 Clostridium difficile toxin A,9 and Streptococcus
pneumoniae protein SpsA.10–12 Interferon-c (IFN-c),13
TNF-a,14 IL-415 and IL-1b16 can up-regulate the releaseof SC into the culture supernatant of the human colonicadenocarcinoma cell line, HT-29. Production of SC by
IFN-c, TNF-a, or IL-1b is regulated by IFN-c regulatory
factor-1 (IRF-1) binding to an element in exon 1 of thepIgR gene that is induced by Janus kinase/signal transducer and activator of transcription (STAT) cascade or
NF-jB.17–19 In addition, IL-4 and IFN-c synergisticallyincreased the release of SC in HT-29 cells.15 These data
indicate that pIgR expression is regulated by cytokines.
We hypothesized that the activation of ERK contributes
to the TNF-a–induced pIgR gene expression. Treatmentof HT-29 cells with TNF-a up-regulates the SC production at the transcriptional level, as previously
described.14,20 However, cotreatment of HT-29 cells with
TNF-a and MAPK kinase (MAPKK, also called MEK1)
inhibitor, PD98059, enhanced the TNF-a–induced SC
production. Our data suggest that the production of SC
in HT-29 cells is also regulated by a negative effect that is
involved in ERK activation.

Materials and methods
cDNA
Human pIgR21–23 cDNA was kindly provided by Prof.
P. Brandtzeag (LIIPAT, Institute of Pathology, National
Hospital, University of Oslo, Oslo, Norway). As a probe
for Northern blot analysis, PstI (668-bp) fragments wereused for the detection of pIgR mRNA.

Reagents
Recombinant human TNF-a (specific activity ¼ 2 ·
107 U/mg) was purchased from Genzyme Corp. (Cambridge,MA).
20 -amino-30 -1,4-diamino-2,3-dicyano-1
4-bis[2-aminophenylmethoxyflavone(PD98059),24
thio]butadiene (U0126),25 2-(4-morpholinyl)-8-phenyl4H-1-benzopyran-4-one (LY294002)26 and antibodies
against phosphoERK1/2 (anti-ACTIVE MAPK) andERK1/2 were purchased from Promega Corp. (Madison,
WI).
 2007 Blackwell Publishing Ltd, Immunology, 123, 500–507

Cell culture
A human colonic adenocarcinoma cell line, HT-29, wasmaintained in MaCoy’s 5 A medium (Life Technologies
Inc., Gaithersburg, MD) supplemented with 10% fetal calf
serum (Cansera International Inc., Ontario, Canada),
1 mM glutamine (Life Technologies Inc., Gaithersburg,
MD), amphotericin B, and penicillin/streptomycin (LifeTechnologies Inc., Gaithersburg, MD) at 37 in an atmosphere of humidified 5% CO2. We selected HT-29 cell line
because this cell line has been often used to examined themechanisms of SC production by TNF-a20,30,31,32 and we
considered that HT-29 is a very good pIgR expression
model. In addition and there is no appropriate cell line touse this study. Prior to treatment, cells (1 · 106 cells/ml)
were plated with fresh medium and cultured. On thesecond day after plating, 10 ng/ml of human recombinant
TNF-a, with or without PD98059, U0126, or LY294002,
was added to the medium of the serum-free condition,and cultivation continued for 48 hr. Then, cultured cells
were harvested, and 2 ml of Solution D was added for
RNA extraction.27 The supernatants were also collected
and subjected to enzyme-linked immunosorbent assay(ELISA), as previously described.16,28 All experiments were
performed in triplicate, and differences between means
were calculated using Student’s t-test.

Northern blot analysis
Ten lg of total RNA was mixed with loading buffer {50%formamide, 6% formalin, 1 · Goldberg buffer [40 mM
Na-MOPS pH 72, 5 mM sodium acetate, 05 mM ethylenediamine N,N,N0 ,N0 tetra-acetic acid (EDTA)]}, denatured
at 65 for 15 min, and cooled on ice. Then, RNA waselectrophoresed through a 10% agarose gel containing
6% formalin. After electrophoresis, the gel was neutralized
by incubation with 025 M ammonium acetate solution
for 5 min, and the RNA was transferred onto Hybond-N(Amersham, Little Chalfont, UK). The RNA was fixed tothe membrane by using a UV crosslinker. Before hybridization, the membrane was incubated in prehybridization solution [50% formamide, 065 M NaCl, 5 mM
EDTA (pH 75), 01% sodium dodecyl sulphate (SDS),
01 M piperazine-N,N0 -bis-[2-ethanesulfonic acid] (PIPES)[pH 68], 5· Denhardt’s solution] at 42 for 6 hr. The
hybridization probe was prepared by using a random
prime DNA labelling kit (TAKARA Shuzo Co., Shiga,
Japan) and purified by Sephadex G-50 (Pharmacia Biotech, Uppsala, Sweden). For hybridization, dextran sulfate(Pharmacia Biotech) was added to the prehybridization
solution at a final concentration of 10%. After hybridization, the membrane was washed four times with washingbuffer (2· standard sodium citrate, 01% SDS, 02%
sodium pyrophosphate) at 55 for 30 min and exposed to
X-OMAT film (Kodak, Rochester, NY) at )80.
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N. Takenouchi-Ohkubo et al.
Reverse trranscription–polymerase chain reaction(RT–PCR)
Cells treated with LY294002 and/or TNF-a were collected
and total RNA was extracted by RNeasy (Qiagen, Valencia, CA). After extraction of total RNA, 500 ng of total
RNA was used for cDNA synthesis using SuperScript III
reverse transcriptase (Invitrogen, San Diego, CA) as manufacture’s instruction. Then, 2 ll of synthesized cDNA
was used as a template for RT–PCR. The sequence ofprimer is as following; human pIgR hSC3 50 -TTGTCT
CCCTGACCCTGAAC-30 , hSC4; 50 -CAAATTCCCTGG
AGTTCTCG-30 (product size is 501 bp), b-actin BACT F
4TH; 50 -TTCCAGCCTTCCTTCCTGG-30 , b-actin BACTR 4TH; 50 -TTGCGCTCAGGAGGAGCAA-30 (product size
is 225 bp).Amplification of human SC and b-action was
performed using HotMasterMix (Eppendorf) as following
condition; 94 for 3 min, then, 94 for 30 s, 60 for 30 s,
72 for 30 s for 35 cycles. Then PCR product was incubated for 8 min at 72 and stored at 4.

Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared by the method as
described previously.20 For preparation of the probe, synthetic oligomers ([jB2, corresponding to )461–440;
50 -GATCGTCGGAGGGGATTCCAGAGCAA-30 , 30 -CAG
CCTCCCCTAAGGTCTCGTTCTAG-50 ) were annealed
and radiolabelled with the Klenow fragment of DNApolymerase I and [a-32P] dCTP (Amersham). For thebinding reaction, 10 lg of nuclear extract was incubatedin 22 ll of a buffer containing 25 mM HEPES (pH 79),2 mM EDTA (pH 80), 50 mM KCl, 10% glycerol, 2 lg ofpoly dI-dC (Pharmacia Biotech), 1% bovine serum albumin (BSA), and 104 counts per minute (c.p.m.) of radiolabelled probe for 30 min at 30. After the reaction, 1 llof loading buffer (5% glycerol, 50 mM EDTA [pH 80],005% bromophenol blue, 005% xylene cyanol) was
added and separated through a 6% native polyacrylamide
gel in TAE buffer (67 mM Tris, 33 mM sodium acetate[pH 60], 10 mM EDTA) at room temperature. The gel
was then dried and exposed to Kodak X-OMAT film at)80 for 12 hr.

Preparation of cell lysate and SDS–Polyacrylamide geleelectrophoresis (PAGE)
For analysis of phosphorylated ERK, HT-29 cells were treated with TNF-a with or without PD98059 at 37 for
15 min and 48 hr. Then, cells were washed with PBS andlysed in cell lysis buffer (20 mM Tris, pH 75, 150 mM NaCl,
025 M sucrose, 5 mM EDTA, 5 mM ethylene glycole bis(b-aminoethyl ether)-N,N,N0 -N0 -tetraacetic acid (EGTA),
05% Triton-X-100, 25 mM NaF, 5 mM Na3VO4, 5 mM
b-glycerophosphate, 15 mM phenylmethylsulphonyl fluo502

ride, 5 lg/ml leupeptin, 10 lg/ml aprotinin),29 and the soluble fraction was collected by centrifugation at 20 000 g at4 for 15 min. Protein concentration was measured by a
Bio-Rad Protein assay kit (Bio-Rad Laboratories, Hercules,
CA). Fifty lg of protein was separated by 10% SDS–PAGE.

Western blot analysis
After electrophoresis, the gel was incubated with transfer
buffer (248 mM Tris, 1925 mM glycine, 005% methanol)for 30 min and transferred onto Immobilon P (Millipore,Bedford, MA) by electroblot. The membrane was incubated in blocking buffer (1% BSA in TBST [20 mM TrisHCl pH 75, 150 mM NaCl, 005% Tween 20]) at room
temperature for 12 hr. Detection of MAPKs was performed
using anti-active MAPK antibody as a primary antibody,
donkey anti-rabbit IgG conjugated with alkaline phosphatase as a secondary antibody, and CDP-Star reagent for signal detection (Amersham). For reprobing, the membrane
was incubated with stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 625 mM Tris-HCl, pH 68) at 50 for
30 min and washed twice with TBST at room temperaturefor 10 min. Then, the membrane was incubated withblocking buffer at room temperature for 16 h and subjected to immunodetection using anti-ERK1/2 antibody.

Results
Production of SC by TNF-a is enhanced by PD98059
To elucidate whether the MAPK is involved in TNF-a signalling and SC production, HT-29 cells were incubatedwith TNF-a for 48 h with 0, 5, 25, or 50 lM of PD98059.
Then, ELISA was performed on culture supernatants
to quantify the amount of SC. The amount of SC isenhanced by TNF-a as compared to the control, as
described previously (data not shown). This effect wasenhanced significantly by addition of PD98059 (P < 0002
when concentration is 50 lM) (Fig. 1a). The same effect
was also observed by pretreatment of HT-29 with
PD98059 for 30 min followed by stimulation of TNF-afor 48 hr (data not shown). SC production by TNF-a wasalso significantly up-regulated by cotreatment with
U0126, an inhibitor of MEK1/2 (P < 001 when concentration is 1 lM) (Fig. 1b).

Enhancement of SC production by PD98059
is regulated at the transcriptional level
To determine if the enhancement of SC production by
PD98059 is regulated at the transcriptional level or translational level, HT-29 cells were treated with TNF-a and5 lM of PD98059 for 48 hr, and northern blot analysis
was performed. The expression of pIgR mRNA inducedby TNF-a was increased by PD98059 (Fig. 2).
 2007 Blackwell Publishing Ltd, Immunology, 123, 500–507

pIgR regulation by MAPK and PI3K
6

6

4

3

2

4

3

2

1

(b)

TNF-α + PD

10 µM

5 µM

48 hr

PD 5 µM

TNF-α

15 minControl

1 µM

TNF-α + U0126

TNF-α + PD

TNF-α + PD98059

0 µM

50 µM

0

TNF-α

(a)

5 µM

0 µM

0

25 µM

1

TNF-α + PD

PD 5 µM

TNF-α

Control

Figure 1. Production of SC by TNF-a is upregulated by addition of MEK1 inhibitor,
PD98059 (a) or MEK1/2 inhibitor U0126 (b)in a concentration-dependent manner. HT-29
cells were treated with 10 ng/ml of TNF-a and0, 5, 25, or 50 lm of PD98059 (a) or 0, 1, 5,
or 10 lm of U0126 (b) and cultured for 48 hr.
Then, supernatants were subjected to ELISA,
and the amounts of SC were measured.

5

Relative increase (× fold)

Relative increase (× fold)

5

pIgR
pERK
β-actin

ERK

Figure 2. Enhancement of SC by TNF-a and PD98029 is controlled
at the transcriptional level. HT-29 cells were treated with TNF-a and5 lm of PD98059 or 1 lm of U0126 or DMSO for 48 hr. Then, total
RNA was extracted and 10 lg of total RNA was subjected to northern blot analysis using human pIgR cDNA.

ERK is activated by TNF-a in HT-29 and inhibitedby PD98059, a MEK inhibitor
To check whether TNF-a-activated ERK1/2 was inhibitedby PD98059, HT-29 cells were treated with TNF-a for
15 min and 48 hr with or without PD98059. Cell lysates
were collected, and phosphorylated ERK1/2 was detectedby western blotting analysis. When HT-29 cells were treated with TNF-a for 15 min, both ERK1 and ERK2 were
phosphorylated as previously reported33 and after 48 hr,
the amount of phosphorylated ERK1/2 was decreased.
Activation of ERK1/2 by TNF-a was completely inhibitedby treatment with 5 lM of PD98059 (Fig. 3). The amount 2007 Blackwell Publishing Ltd, Immunology, 123, 500–507

Figure 3. Phosphorylation of ERK1/2 by TNF-a is blocked by 5 lmof PD98059. HT-29 cells were treated with 10 ng/ml of TNF-a and5 lm of PD98059 or DMSO for 15 min. Then, protein was extracted
and phosphorylated ERK1/2 was detected by Western blot using
antiactive ERK antibody. To check the amount of total ERK1/2,
anti-ERK1/2 antibody was used.

of ERK protein did not change by treatment of TNF-a
with or without PD98059 (Fig. 3).

SC production by TNF-a is inhibited byphosphatidylinositol-3-kinase (PI3K) inhibitor
LY294002
PI3K is also activated by extracellular signals such as
growth factors, cytokines, integrin-mediated signals, and
503

iv
e

at

eg

+
LY

LY

N

Fα
TN

C

on

tro

l

TN
Fα

1·2

co
nt
Po
ro
si
l
tiv
e
co
nt
ro
l

N. Takenouchi-Ohkubo et al.

1·0
pIgR

Relative increase (× fold)

0·8β-actinFigure 5. TNF-a mediated SC production inhibited by LY294002 isregulated at the transcriptional level. HT-29 cells were treated with
LY294002 and TNF-a for 48 hr. Then total RNA was extracted and
RT–PCR was performed to detect pIgR mRNA. For positive control
of pIgR, 2 ng of full length pIgR cDNA was used.

0·6

0·4

free SC constitutively, suggesting that LY294002 inhibited
mainly constitutively expressed pIgR mRNA.
0·2

Involvement of NF-jB in the enhancement of SC
production by PD98059 and inhibition by Ly294002

50 µM

25 µM

5 µM

0 µM

0·0

TNF-α + LY294002

Figure 4. SC production by TNF-a is inhibited by LY294002.HT-29
cells were treated with 10 ng/ml of TNF-a with 0, 5, 25, or 50 lm of
LY294002 for 48 hr. Then, supernatants were subjected to ELISA,
and the amounts of SC were measured.

other survival signals.34 To elucidate whether PI3K isinvolved in TNF-a–induced SC production, HT-29 cells
were costimulated with TNF-a and LY294002 for 48 hr,
and then the amount of SC protein in culture supernatants was quantified. The production of SC was inhibitedby PI3K inhibitor as previously reported35,36 at any concentration, especially at the 50 lM concentration (Fig. 4).

Inhibition of SC production by LY294002 is regulated
at the transcriptional level
It has been indicated that PI3K inhibitor inhibit transcytosis of pIgR.35,36 To check whether inhibition of pIgR
by LY294002 is regulated at the transcriptional or posttranscriptional level, RT–PCR was performed. As weshowed the decrease of SC production by LY294002,
expression level of pIgR mRNA was also decreased ascompared with TNF-a stimulation. This data suggest that
inhibition of SC production by LY294002 was regulated
at the transcriptional level (Fig. 5). Treatment of HT-29
with only LY294002 completely inhibited the expressionof SC mRNA as compared with control, which express
504

Treatment of glioblastoma cell line T98G with PD98059
and IL-1b augmented NF-jB activation.37 NF-jB is an
important transcription factor for the pIgR gene.20,30–32
We checked whether the enhanced SC production by
PD98059 is via enhanced NF-jB activation by using a gel
shift assay to detect the NF-jB bound upstream of thepIgR gene (jB2). The amount of NF-jB binding to thejB2 site did not increase after PD98059 treatment (Fig. 6).
We also checked whether inhibition of PI3K influences theeffect of NF-jB activation. HT-29 cells were treated with
10 ng/ml of TNF-a with or without LY294002 for 90 min,and then nuclear extracts were prepared and the gel shift
assay was performed using jB2 probe. Co-treatment of
HT-29 cells with TNF-a and LY294002 did not change thebinding of NF-jB to jB2 (Fig. 6).

Inhibition of PI3K by TNF-a is independentof ERK activation
To determine if inhibition of PI3K affected ERK activation, HT-29 cells were treated with 50 lM of LY294002
for 15 min. Cell lysates were applied to western blots.
The amount of phosphorylated ERK was unchanged bycotreatment with TNF-a and LY294002 (Fig. 7).

Discussion
In IBD such as Crohn’s disease and ulcerative colitis, thenumber of TNF-a-producing cells is increased in thesmall intestine.2 Some treatment strategies for IBD target
TNF using anti-TNF monoclonal antibody (infliximab)38
or MAPK signalling pathway inhibitor (CNI-1493).39 On
 2007 Blackwell Publishing Ltd, Immunology, 123, 500–507

TNF-α + LY

LY

TNF-α + PD

PD

TNF-α

ControlpIgR regulation by MAPK and PI3K

NF-κB

(a)
1

0·5

0
(b) Control

TNF-α

PD

TNF-α
+ PD

LY

TNF-α
+ LY

TNF-α + LY

LY50 µM

TNF-α

Control

Figure 6. Binding of NF-jB to jB2 is not affected by PD98059 andLY294002. (a) HT-29 cells were treated with 10 ng/ml of TNF-a and5 lm of PD98059 or 50 lm of LY294002 for 90 min, and thennuclear protein was extracted. Ten lg of nuclear protein was used
for the gel shift assay. (b) Densitometric analysis of gel shift assay
using Image J software. Y-axis shows the density of band comparedto TNF-a stimulation (¼ 1) as a control.

pERK

ERK
(a)
1·5

1

0·5

0
(b)

Control

TNF-α

LY

TNF-α + LY

Figure 7. Activation of ERK by TNF-a was not altered by LY294002.
(a) HT-29 cells were treated with 10 ng/ml of TNF-a and 50 lm of
LY294002 for 15 min. Then, protein was extracted and phosphorylated ERK1/2 was detected by Western blot using antiactive ERK
antibody. (b) Densitometric analysis using Image J software.

 2007 Blackwell Publishing Ltd, Immunology, 123, 500–507

the other hand, the expression levels of pIgR and S-IgA
were decreased in IBD.40 In lung epithelial cells, expression of SC is reduced in chronic obstructive pulmonary
disease41 and in this condition SC is cleaved by serine
protease produced by phorbol myristate-activated polynuclear neutrophil (PMN).42
In this study, we reported that production of SC by
TNF-a was enhanced by MEK1/2 inhibitors (Fig. 1) without the redundant NF-jB activation (Fig. 6). Our datasuggest that MAP kinase signalling pathway inhibitor is abeneficial treatment for IBD because there is a possibility
that the decreased pIgR protein and S-IgA would be elevated and that might enhance the mucosal immunity
without excessive NF-jB activation.
There are several reports about the signalling mechanisms that enhance the expression level of pIgR by various cytokines. When HT-29 cells are treated with IFN-cand IL-4, the expression level of pIgR mRNA is up-regulated, and protein tyrosine kinase is involved in up-regulation of pIgR.43 In addition, the STAT-6 binding site
located at intron 1 is important for the up-regulation ofpIgR gene expression by IL-444 and co-operation of
STAT-6 and tissue-specific factor, hepatocyte NF-1, is also
required.45 PIgR gene expression induced by TNF-a has
also been suggested to be regulated by NF-jB locatedupstream of the transcription initiation site20 and anotherbinding site located at intron 145 is strongly suggested to
be involved. However, signalling mechanisms by TNF-a
were not well elucidated.
ERKs were activated by treatment of HT-29 cells with
TNF-a (Fig. 3), and production of SC by TNF-a wasenhanced by inhibition of ERKs (Fig. 1). These data indicate the existence of a negative regulation pathway by
TNF-a stimulation. In addition, PI3K inhibition by
LY294002 decreased the production of SC by TNF-a(Fig. 4) without NF-jB inactivation (Fig. 6) and ERK
activation (Fig. 7), suggesting that the PI3K pathway positively regulates SC production at the transcriptional level.
As it has been reported that PI3K is involved in transcytosis of pIgR35,36 inhibition of SC production by PI3K
inhibitor is controlled at the transcriptional level (Fig. 5).
We could not detect SC production by ELISA from thecontrol sample but could detect by RT–PCR, probably
depending on the different sensitivities of detection
system. These data suggest that PI3K is involved in trancytosis of pIgR in the HT-29 cells as previously indicated8,35,36 and our data confirm previous data. Because
we have indicated that expression of pIgR mRNA was notcompletely inhibited by NF-jB inhibitor20 it is possiblethat there is also an NF-jB-independent pathway. In
bronchial epithelial cells, up-regulation of SC production
by activated PMN was also inhibited by NF-jB and p38
inhibitor. However, activation of NF-jB and p38 is independent of SC production by serine proteinase, whichmeans that SC cleavage and expression are independently505

N. Takenouchi-Ohkubo et al.
regulated.42 Our data indicate that there are several kindsof signalling pathways, such as NF-jB-dependent
and NF-jB-independent pathways, which are involved in
SC production by TNF-a. Our data also indicate theexistence of negative and positive regulators of the
NF-jB-independent pathway in HT-29 cells. Further
investigations are needed to elucidate the complicatedmechanisms of signalling pathways for SC production.

Acknowledgements
Financial support was obtained from the Nihon University Research Grant for Assistants and Young Researchers(2003), the Grant from the Ministry of Education, Culture, Sports, Science and Technology to promote multidisciplinary research projects, and Academic Frontier
Project for Private Universities: matching fund subsidy
from MEXT (to Dr Hiroki Nagase).

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o mw w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Phosphorylation of Extracellular Signal-Regulated Kinase in
medullary and upper cervical cord neurons following noxious
tooth pulp stimulation
Kohei Shimizu a , Masatake Asano b , Junichi Kitagawa c,e , Bunnai Ogiso a,d , Ke Ren f ,Hidero Oki g , Mitsuhiko Matsumoto g , Koichi Iwata c,e,h,⁎aDepartment of Endodontics, School of Dentistry, Nihon University, 1-8-13 Kandasurugadai, Chiyoda-ku, Tokyo 101-8310, Japan
Department of Pathology, School of Dentistry, Nihon University, 1-8-13 Kandasurugadai, Chiyoda-ku, Tokyo 101-8310, Japanc
Department of Physiology, School of Dentistry, Nihon University, 1-8-13 Kandasurugadai, Chiyoda-ku, Tokyo 101-8310, Japand
Division of Advanced Dental Treatment, Dental Research Center, Nihon University School of Dentistry, Tokyo 101-8310, Japane
Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry, Tokyo 101-8310, Japanf
Department of Biomedical Sciences, University of Maryland Dental School, Baltimore, MD 21201, USAg
Department of Oral and Maxillofacial Surgery, School of Dentistry, Nihon University, 1-8-13 Kandasurugadai,
Chiyoda-ku, Tokyo 101-8310, JapanhDivision of Applied System Neuroscience Advanced Medical Research Center, Nihon University Graduate School of Medical Science,
30-1 Ohyaguchi-Kamimachi Itabashi, Tokyo 173-8610, Japanb

A R T I C LE I N FO

AB S T R A C T

Article history:

The phosphorylated Extracellular Signal-regulated Kinase (pERK) and Fos expression andAccepted 5 December 2005

masticatory muscle activity were analyzed in rats with capsaicin-induced acute

Available online 25 January 2006

inflammation of the tooth pulp in order to clarify the role of the spinal trigeminal nucleus
and upper cervical spinal cord in tooth pulp pain. Digastric and masseteric muscle activities

Keywords:

were significantly increased following capsaicin injection into the molar tooth pulp but not

MAPK

after vehicle treatment. The pERK-like immunoreactive (LI) neurons were observed in the

ERK

subnuclei interpolaris–caudalis transition (Vi/Vc) zone, the paratrigeminal nucleus (Pa5) andFos

the superficial laminae of the caudal Vc/C2 zone. The pERK expression was detected as early

Tooth pulp

as 2 min and peaked at 5 min after capsaicin or vehicle injection. The pERK expression in the

Capsaicin

Vi/Vc zone and Pa5 was bilateral, whereas it was predominantly ipsilateral in the caudal Vc/

Spinal trigeminal nucleus

C2 zone. The capsaicin treatment of the whisker pad produced pERK expression in the
rostro-caudal middle portion of the ipsilateral Vc, but small number of pERK-LI cells wereobserved after vehicle treatment. The pERK expression was similar in the Vi/Vc zone
following capsaicin injection into the upper or lower molar tooth pulp, whereas the pERK
expression was in the lateral portion of the caudal Vc/C2 zone after upper molar injection
and restricted to the medial portion of the Vc/C2 zone after the lower molar capsaicin. These
data were confirmed with Western blots. There were differences in the distribution of Fos
protein-like immunoreactive (LI) cells and pERK-LI cells following tooth pulp stimulation.
After capsaicin application into the upper molar tooth pulp, no pERK-LI cells were observed⁎ Corresponding author. Fax: +81 3 3219 8341.
E-mail address: iwata-k@dent.nihon-u.ac.jp (K. Iwata).
0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2005.12.040

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in the ventral part of the Vi/Vc zone, whereas many Fos protein-LI cells were expressed inthis region. The difference in the distribution pattern of pERK- and Fos protein-LI cells in the
Vi/Vc zone suggests their differential temporal expression profiles after capsaicin. The
present findings suggest that tooth-pulp-driven neurons in the spinal trigeminal nucleus
are involved in tooth pulp pain through activation of the intracellular signal transduction
pathway that involves earlier ERK phosphorylation and subsequent Fos expression.
© 2005 Elsevier B.V. All rights reserved.

1.

Introduction

The Extracellular-Signal-regulated Kinase (ERK) is one of themitogen-activated protein kinases (MAPKs) activated bycalcium influx in the dorsal root ganglion (DRG) and dorsal
horn (DH) neurons (Ji et al., 1999, 2002; Kawasaki et al., 2004).
A variety of peripheral stimuli activate MAPKs pathways (Daiet al., 2002; Ji et al., 2002; Liu et al., 2004; Obata et al., 2004a,b).
Phosphorylation of ERK (pERK) is known to be induced as aresult of neuronal activation. Specifically, it has beenreported that C-fiber stimulation induces pERK expression
in the DH neurons but not low threshold A-fiber stimulation(Ji et al., 1999). It has been also reported that the pERK
expression peaked in DRG and DH neurons within 10 min
after noxious stimulation (Dai et al., 2002; Liu et al., 2004;
Wang et al., 2004). The pERK expression in DH neurons was
also modulated by NMDA receptor antagonist (Ji et al., 1999,
2002). These data suggest that pERK is a potential marker ofthe excitability of DH nociceptive neurons following noxious
stimulation of peripheral structures, which occurs at theearly period after stimulation. The digastric and massetericmuscles are known to be activated by the noxious stimulation of the tooth pulp as well as a variety of trigeminal
structures (Park et al., 2001; Sunakawa et al., 1999). The EMG
activity was reported to be elicited just after noxious
stimulation of the tooth pulp and lasting for a while(Sunakawa et al., 1999). The time-course of the EMG activity
was similar to that of pERK expression. Therefore, it is very
convenient to compare the EMG activity and pERK expressionin order to study the change in neuronal activity after
trigeminal noxious stimulation.
A large number of nociceptive neurons in the subnucleus
caudalis (Vc) of the spinal trigeminal nucleus (Sp5) areresponsive to a variety of stimuli applied to the tooth pulp
(Chattipakorn et al., 2001, 2002; Hu and Sessle, 1984; Hu et al.,
1981; Iwata et al., 1998; Oakden and Boissonade, 1998; Sessle,
1987; Sessle and Greenwood, 1976; Sessle et al., 1986; Sugimoto
et al., 1998). The Vc has been known as an important nucleus
relaying tooth pulp sensory information to the higher central
nervous system (CNS). Electrical stimulation of the tooth pulp
induces a large number of Fos protein-LI cells in the transitionzone between the subnuclei interpolaris and caudalis (Vi/Vczone) and the caudal Vc and the upper cervical cord (Vc/C2zone). These two zones play an important role in conveying
sensory information of the tooth pulp (Iwata et al., 1998).
Electrophysiological studies have reported that most Vc
neurons responsive to tooth pulp stimulation (tooth-pulpdriven neurons) receive noxious and/or non-noxious cutaneous inputs from the jaw and orofacial regions (Sessle, 1987;
Sessle and Greenwood, 1976; Sessle et al., 1986). Thus, toothpulp-driven neurons in the Vc have functional roles in relaying

trigeminal cutaneous information as well as tooth pulp input.
It is still unknown how these two CNS regions are involved inprocessing tooth pulp noxious information. In the present
study, we focused on these two regions to clarify the functional
differences of tooth-pulp-driven neurons activated by capsaicin injection. It has been known that Fos is a marker of dorsal
horn neuronal activation following peripheral noxious stimulation. However, the Fos analysis is not adequate to study theevent occurring at the early time period after noxious
stimulation of peripheral structures. A variety of chemicals
are known as the possible peripheral irritants to activate
peripheral nociceptors. However, capsaicin is one of theirritants whose peripheral mechanism is well known, capsaicin is dominantly involved in an acute activation of C-fiber
afferents (Caterina et al., 1997). Thus, the pERK expression in
the Sp5 and upper cervical cord was analyzed in rats receiving
capsaicin treatment of the tooth pulp in order to clarify the
distribution pattern of active neurons in the medulla and theupper cervical cord at the early period after capsaicinstimulation, and the results compared to that of Fos expression
and that following cutaneous capsaicin injection into theregion of the whisker pad.

2.

Results

The digastric and masseteric electromyographic (EMG) muscle
activities were significantly increased following capsaicin
injection into the upper molar tooth pulp as illustrated in Fig.
1. The EMG activities of these two muscles were simultaneously increased and lasted for about 3–5 min after capsaicin
injection. In general, the capsaicin-evoked digastric EMG
activity was much larger and longer lasting as compared tothe masseteric EMG (Fig. 1B). There was no change in EMG
activities following the vehicle injection into the molar tooth
pulp (Fig. 1C).
The pERK-like immunoreactive (LI) neurons were observed in the dorsal portion of the Vi/Vc zone and Pa5
bilaterally 5 min after capsaicin injection into the upper
molar (Figs. 2A–E and 3). In the caudal Vc/C2 zone, pERK-LI
neurons were mainly found in the lateral portion of theipsilateral superficial laminae, and there were only a few
pERK-LI cells in the contralateral Vc/C2 zone (Figs. 2C and F).
The increase in pERK activity in the Vi/Vc zone (Pa5 wasincluded in this region) and Vc/C2 zone was verified with
Western blot analysis following capsaicin injection into thetooth pulp as compared to vehicle-treated rats (Figs. 2Ga andb). The pERK and total ERK immuno-products exhibited as
two 42 kDa and 44 kDa bands on Western blot (Fig. 2G). The
pERK expression was different between the Vi/Vc zone + Pa5
and Vc/C2 zone. The 42 kDa band was expressed in the Vi/Vc

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101

Fig. 1 – Digastric and masseter muscle EMG activities ipsilateral to capsaicin or vehicle injection. (A) Typical responses of
EMG activities recorded from digastric (Dig) and masseter (Mass) muscles following capsaicin injection into the upper molar
tooth pulp. (B) Percent change in Dig and Mass muscle activities following capsaicin injection into the upper molar tooth
pulp (n = 5). (C) Percent change in Dig and Mass muscle activities following vehicle injection into the upper molar tooth pulp.
cap: capsaicin, veh: vehicle (LSD test).

zone + Pa5 bilaterally, whereas it was only observed in theipsilateral Vc (Fig. 2G). This is consistent with the immunohistochemistry results (Figs. 2A–F). There were no differencesin the total ERK expression between the Vi/Vc zone + Pa5 andVc/C2 zones and also no differences between capsaicin andvehicle-treated rats.
The camera-lucida drawings of pERK-LI cells are illustrated
in Fig. 3. The pERK-LI cells were basically restricted to the
nucleus and are represented as small dots in each section.
Following capsaicin injection into the upper molar, a large
number of pERK-LI cells were expressed in the dorsomedial
portion of the Vi/Vc zone bilaterally. On the other hand, pERKLI cells showed relatively wider distribution in the Vi/Vc zone
following lower molar injection. We also observed a large
number of pERK-LI cells in the rostro-caudally middle portionof the ipsilateral Vc following capsaicin injection into thewhisker pad region. A few pERK-LI cells were expressed in thecontralateral Vc after whisker pad injection. In the Vc/C2 zone,
the distribution patterns of pERK-LI cells were different
between the injections into the upper and lower molars andwhisker pad. The pERK-LI cells were restricted in the lateral
portion of the ipsilateral Vc/C2 zone after capsaicin injection
into the upper molar. On the other hand, pERK-LI cells were
distributed in the medial half of the ipsilateral Vc/C2 zone
following lower molar injection. Only a few pERK-LI cells wereobserved in the ipsilateral Vc/C2 zone following capsaicin
injection into the whisker pad region. In the Pa5, a small

number of pERK-LI cells were expressed bilaterally after
capsaicin injection into the molar tooth pulps. The pERK-LI
cells in the rostral Pa5 were significantly larger in the ipsilateralside as compared to contralateral side (Fig. 4A). However,
distribution pattern of pERK-LI cells in the Pa5 through rostrocaudal extension was similar between ipsilateral and contralateral sides. Almost no pERK-LI cells were expressed in thecontralateral Vc following whisker pad capsaicin injection.
There were also pERK-LI cells in the Vi/Vc zone, Pa5 and Vc/C2zone following vehicle injection into the molar tooth pulps.
Although the distribution patterns of pERK-LI cells after
vehicle injection into the tooth pulp were similar to that after
capsaicin treatment, there was a significant difference in thenumber of pERK-LI cells (Fig. 4).
The rostro-caudal distribution of pERK-LI cells following
capsaicin or vehicle treatment of the tooth pulp is summarized in Fig. 4. Following capsaicin injection into the upper
molar tooth pulp, the pERK-LI cells were expressed with two
peaks along the rostro-caudal extent of the Vc. One peak
was in the Vi/Vc zone about 0–0.7 mm caudal to the obex,
and the other peak was in the Vc/C2 zone at about 6.5 mm
caudal to the obex. There were no distribution differences in
pERK-LI cells between the ipsilateral and contralateral sidesof the Vi/Vc zone following capsaicin injection into theupper molar tooth pulp (Fig. 4A, p N 0.05, Student's orWelch's test). On the other hand, the number of pERK-LI
cells was significantly larger in the ipsilateral side than that

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Fig. 2 – Capsaicin-induced pERK-LI cells in the medulla and upper cervical spinal cord. (A) Ipsilateral Vi/Vc zone. (B) IpsilateralPa5. (C) Ipsilateral Vc/C2 zone. (D) Contralateral Vi/Vc zone. (E) Contralateral Pa5. (F) Contralateral Vc/C2 zone. (G) Western
blot illustrating the levels of pERK and total ERK in the Vi/Vc zone + Pa5 and Vc/C2 zone in the rat with capsaicin stimulation of
the tooth pulp (a) and without capsaicin stimulation (b). Vc: trigeminal spinal nucleus caudalis, Pa5: paratrigeminal nucleus,
C2: second cervical spinal cord. ipsi: ipsilateral, cont: contralateral.

of contralateral side in the Vc/C2 zone after capsaicin
injection into the upper molar tooth pulp (Figs. 4A and C,
p b 0.05, Student's or Welch's test). The pERK-LI cells werealso seen bilaterally in the Pa5. A small number of pERK-LI

cells were expressed in the Vi/Vc zone, Pa5 and Vc/C2 zone
after vehicle injection into the tooth pulp, but the number ofpERK-LI cells was significantly smaller as compared to thatof the capsaicin-treated rats.

Fig. 3 – Camera-lucida drawings of pERK-LI cells in the medulla and upper cervical spinal cord following capsaicin injection
into the tooth pulp or whisker pad region. Each dot indicates one pERK-LI cell. Solid arrows: Pa5 regions, open arrows:
Vi/Vc zone, small arrows: Vc/C2 zones. The negative numbers on the left indicate the distance caudal to the obex.

BR A I N R ES E A RC H 1 0 7 2 ( 2 00 6 ) 9 9 –1 09

103

Fig. 4 – Rostro-caudal distribution of pERK-LI cells in the medulla and upper cervical spinal cord. The number of pERK-LI
cells was plotted against the distance caudal the obex. (A) Data from the capsaicin-treated rats (n = 5). (B) Data from thevehicle-treated rats. (C) Laminar distribution of pERK-LI cells in the Vc/C2 zone of the capsaicin-treated rats. (D) Laminar
distribution of pERK-LI cells in the Vc/C2 zone of the vehicle-injected rats. Sp5: spinal trigeminal nucleus (ipsi vs. cont: Student's
or Welch's test, cap vs. veh: Newmann–Keuls test).

The time-course of the changes in pERK expression following
capsaicin treatment of the upper molar tooth pulp is illustrated
in Fig. 5. We observed significant increase in EMG activity within

Fig. 5 – Time-course of the change in the number of pERK-LI
cells in the Vi/Vc zone (A), Pa5 (B) and Vc/C2 zone (C) following
capsaicin injection into the upper molar tooth pulp (n = 5 pertime point). naive: rats without any stimulation, veh: rats
with vehicle injection into the tooth pulp (naive vs. cap:
Dunnett's test, ipsi vs. cont: Student's or Welch's test).

5 min following capsaicin injection into the tooth pulp withsimilar time-course in pERK expression as illustrated in Figs. 1
and 5. The pERK expression started at 2 min and peaked at 5
min after capsaicin injection. After the 5 min time point, thenumber of pERK-LI cells gradually decreased and returned to thecontrol level at 2 h after capsaicin injection (Fig. 5). The changes
in the number of pERK-LI cells showed a similar time-courses inboth sides of the Vi/Vc zone (Fig. 5A). The time-course of thechange in the number of pERK-LI cells in the Pa5 following
capsaicin treatment of the upper molar tooth pulp was similar tothat of the Vi/Vc zone (Fig. 5B). The changes in the number ofpERK-LI cells showed a similar time-course following capsaicin
treatment of the tooth pulp in the ipsilateral Vc/C2 zone (Fig. 5C).
The comparison of distribution patterns of pERK-LI cells
between the tooth pulp injection and whisker pad injection is
illustrated in Fig. 6. Following capsaicin injection into thewhisker pad region, a significantly larger number of pERK-LI
cells were seen in the ipsilateral Vi/Vc zone and Vc/C2 zone ascompared to the contralateral side (Fig. 6). On the other hand,the number of pERK-LI cells was not different between
ipsilateral and contralateral sides in the Vi/Vc zone andipsilateral dominant in the Vc/C2 zone after capsaicin treatment of the molar tooth pulp (Fig. 6). We also studied thecomparison between pERK expression and Fos expression in
the rats with capsaicin treatment of the upper tooth pulp.
A large number of Fos protein-LI cell were observed in thedorsal and ventral parts of the Vi/Vc zone and Pa5 bilaterally,
and in the Vc/C2 zone with ipsilateral dominant 1 h after
capsaicin treatment of the upper tooth pulp (Fig. 7). We could
observe the significant expression of Fos protein-LI cells in
the rats with vehicle treatment (Fig. 7B). The distributiondifference in Fos protein- and pERK-LI cells was observed in

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Fig. 6 – The number of pERK-LI cells in the Vi/Vc zone, Pa5 and Vc/C2 following capsaicin injection into the whisker pad (A) andthe expression of pERK-LI cells in the rats with capsaicin injection into the upper molar tooth pulp (B). cap: capsaicin,
veh: vehicle (ipsi vs. cont: Student's or Welch's test).

the ventral portion of the Vi/Vc zone as indicated by thearrows in Fig. 7B. No pERK-LI cells were expressed in theventral portion of the Vi/Vc zone following tooth pulp
capsaicin treatment (Fig. 3).

3.

Discussion

Tooth pulp afferents project to the Sp5 and were somatotopically arranged in the Sp5 complex (Pertovaara et al., 1987;
Sessle, 1987; Shigenaga et al., 1986, 1989). Tooth pulp afferents
contain both high and low threshold nerve fibers (Jyvasjarvi
and Kniffki, 1992; Narhi et al., 1992). It is well known that thesensory innervation of tooth pulp is very rich, and it consists
almost purely of nociceptive Aδ- and C-fibers and of very
scarce amount of fast conducting Aβ-fibers (Byers, 1984). The
functional role of these pulpal Aβ-fibers is unclear, and, forexample, they do not respond to non-noxious mechanicalstimulation of the intact tooth (Narhi et al., 1992). More recent

studies and detailed comparisons of the response characteristics of pulpal nociceptive Aδ- and Aβ-fibers indicated thatthey most likely belong to the same functional group (Byers
and Narhi, 1999; Narhi et al., 1994).
Aδ and C-fibers in the tooth pulp are known to be involved
in tooth pulp pain. Aδ-fibers in the tooth pulp are activated bya variety of natural stimuli applied to the tooth (Addy and
Pearce, 1994). On the other hand, the tooth pulp C-fibers
respond to the noxious stimulation of the pulp such as
chemical and thermal stimuli (Narhi et al., 1992; Sessle,
1987). Aδ-fibers are involved in acute non-inflamed pain,
whereas C-fibers are involved in tooth pulp inflamed pain. It is
still unclear, however, how these two different types of tooth
pulp afferents contribute to activation of CNS neurons that
related to tooth pulp pain. Recent reports indicate that the Vi/
Vc zone, Pa5 and upper cervical cord have differential roles inprocessing noxious information in the trigeminal system(Chattipakorn et al., 2001, 2002; Iwata et al., 1998, 2004). Fos
expression is induced at about 1 h after noxious stimulation ofFig. 7 – Fos expression in the Vi/Vc zone, Pa5 and Vc/C2 zone following capsaicin injection into the upper molar tooth pulp.
(A) Photomicrographs of Fos protein-LI cells in the Vi/Vc zone, Pa5 and Vc/C2 zone. (B) Camera-lucida drawings of Fos
protein-LI cells in the medulla and upper cervical spinal cord 1 h after capsaicin injection into the upper molar tooth pulp.
Arrows indicate the ventral region of Vi/Vc zone.

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peripheral structures (Tokunaga et al., 1995). On the other
hand, pERK is expressed within 10 min following noxious
peripheral stimulation (Ji et al., 2002). The pERK expression
can be used as a measure of the early event which occurred in
dorsal horn neurons after noxious peripheral stimulation.

3.1.

Changes in digastric and masseteric muscle activities

The digastric and masseteric muscles are activated by
noxious stimulation of the tooth pulp (Park et al., 2001;
Sunakawa et al., 1999). The EMG activity recorded from thesemuscles is a good indicator of noxious responses in thetrigeminal system. We observed a significant increase in EMG
activity of these muscles following capsaicin injection into themolar tooth pulp, but not following vehicle injection (Fig. 1).
The increase in EMG activity lasted for about 3–5 min,although a significant difference was only detected within 1
min. The time-course change in EMG activity was similar tothat of pERK expression following capsaicin treatment of the
tooth pulp. These data strongly suggest that the increase in
EMG activity and pERK expression after the capsaicin application to the tooth pulp should be a result of the activation of
intra-pulpal high threshold afferents including C-fibers and an
acute tooth pulp inflammation.

3.2.

pERK-LI cells in the Vi/Vc zone and Pa5

It has been reported that glial cells in the spinal dorsal horn(DH) express pERK following peripheral inflammation (Zhuang
et al., 2005). It may be possible that pERK is expressed in the
glial cells of Vi/Vc and Vc/C2 zones after tooth pulp inflammation such as spinal DH. The expression of pERK in the glial
cells is observed 1–2 weeks after inflammation, and those with
pERK are distributed in the wide area of DH in both sides(Zhuang et al., 2005) The Vi/Vc and Vc/C2 neurons with pERK
following capsaicin treatment of the tooth pulp were localized
in the superficial laminae and were also ipsilateral dominant.
These data strongly suggest that pERK-LI cells we observed
after capsaicin tooth pulp treatment are expressed in the Vi/
Vc and Vc/C2 neurons but not in glial cells. We observed alarge number of pERK-LI cells in the dorsal portion of the Vi/Vczone and Pa5 bilaterally following capsaicin injection into theupper or lower molar tooth pulp. Previous HRP tracing studies
have reported that tooth pulp afferents were somatotopicallyarranged in the cat trigeminal nuclei (Sp5) (Shigenaga et al.,1986, 1989). Upper tooth pulp afferents project to the ventral
portion of the Vc, whereas lower tooth pulp afferents were
distributed in the dorsal portion of Vc. The distribution areasof pERK-LI cells within Vc and Pa5 following capsaicin
injection into the tooth pulp were similar in the upper andlower molar tooth as illustrated in Fig. 3. The pERK-LI cells
were not observed in the ventral portion of the Vi/Vc
transition zone where Fos protein-LI cells were expressedfollowing capsaicin injection into the upper molar tooth pulp
(Fig. 7). The distribution area of pERK-LI cells in Vi/Vc zone andPa5 was different from the results of Fos study where Fos
protein-LI cells were bilaterally represented in the ventral Vi/
Vc zone and Pa5. These results indicate that the Vi/Vc zone
and Pa5 regions lack somatotopic organization for trigeminal
nociception. It has been reported that Fos protein-LI cells are105

expressed in spinal DH 0.5–1.0 h following noxious stimulationof the skin, whereas pERK-LI cells are expressed within 10 min(Ji et al., 2002; Tokunaga et al., 1995). The time-course
difference in expression between pERK and Fos protein may
be reflected in the distribution differences of positive neurons
in the Vi/Vc zone. It is probable that pERK activation is anearlier event in tooth pulp nociception in the region ascompared with that of Fos.
It has been known that the topographical organization of
nociceptive neurons in the Vc is an important factor in thediscrimination of tooth pulp pain (Dubner et al., 1989; Priceand Dubner, 1977). On the other hand, the capsaicin treatmentof the whisker pad produces a large number of pERK-LI cells in
the restricted area of the ipsilateral Vc. This region corresponds to the whisker pad primary afferent projection area in
Vc (Takemura et al., 1991). These suggest that pERK-LI cells
expressed in the Vi/Vc zone and Pa5 following tooth pulp
stimulation would not be involved in the sensory discriminative aspect of tooth pulp pain, whereas those expressed after
whisker pad stimulation may have a function to discriminate
nociception of the facial skin. Some previous studies reported
that Fos expression in Vi/Vc and Pa5 following temporomandibular inflammation was strongly affected by vagal nerve
transection (Bereiter et al., 2002). These data and the present
findings suggest that the Vi/Vc and Pa5 neurons are involvedin autonomic regulation as well as trigeminal nociception.

3.3.

pERK expression in the Vc/C2 zone

We observed a significant increase in pERK expression in
the Vc/C2 ipsilaterally, whereas a few pERK-LI cells wereobserved in the contralateral Vc/C2 zone following capsaicin
injection into the molar tooth pulp. We also observed many
Fos protein-LI cells in this region 1 h after capsaicin injection
into the upper molar tooth pulp. It has been reported that
Fos protein-LI cells are strongly expressed in the ipsilateral
Vc/C2 zone following TMJ inflammation, parotid glandinflammation or tooth pulp stimulation (Hathaway et al.,
1995; Nomura et al., 2002; Ogawa et al., 2003; Zhou et al.,1999). Nociceptive neurons in the Vc/C2 zone may receive
direct inputs from the TMJ, parotid gland or tooth pulp andexhibit increased neuronal activity following TMJ, parotid
gland or tooth pulp inflammation. It is well known thatperipheral inflammation produces an extensive increase in
the excitability of small diameter primary afferent nerve
fibers, resulting in hyperexcitability of the second orderneurons (Chiang et al., 1998; Hylden et al., 1989; Zhou et al.,1999). We observed that the pERK-LI cells were differentiallyarranged in the Vc/C2 zone following upper or lower molar
tooth pulp stimulation. Capsaicin injection into the upper
tooth pulp induced a large number of pERK-LI cells in thelateral portion of the Vc/C2 zone, whereas capsaicin
injection into the lower tooth pulp resulted in pERK-LI cells
in the medial portion of Vc/C2 zone. A small number ofpERK-LI cells were expressed after capsaicin injection into
the whisker pad. The difference in the topographicalarrangement of pERK-LI cells in Vc/C2 zone following
different tooth pulp stimulation was consistent with topography of trigeminal afferents and a role for tooth pulp
nociceptive neurons to discriminate tooth pulp pain.

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It has been reported that the tooth-pulp-driven neurons arealso recorded from the spinal trigeminal subnucleus oralis(Vo) (Park et al., 2001). Previous anatomical tracing studies
have shown that there are direct projection fibers from thetooth pulp to the Vo as well as Vc in cats (Shigenaga et al.,1989). There may be two possible mechanisms to activate Vo
neurons by tooth pulp stimulation as follows: the Vo neurons
are directly activated by tooth pulp primary afferents or are
activated by the afferent inputs through Vc neurons. We could
not observe any pERK-LI cells in Vo following capsaicinstimulation of the tooth pulp. It is likely that Vc neurons are
directly activated by capsaicin injection into the tooth pulp at
the early time period following tooth pulp stimulation and Vo
neurons receive high threshold tooth pulp inputs originatedfrom the pulpal C- and Aδ-fiber afferents through Vi/Vc and/or
Vc/C2 neurons.
We also observed a large number of pERK-LI cells in the Vi/
Vc zone, Pa5 and Vc/C2 zone following vehicle injection into
the tooth pulp. The dental paper points were used for
application of vehicle into the tooth pulp. The pERK expression following vehicle injection into the pulp may beoriginated from the direct mechanical stimulation of the
tooth pulp.

3.4.

Functional significance of pERK in tooth pulp pain

The ERK activation is known as an important step in the
MAP kinase cascade involved in the intracellular signaltransduction (Ji et al., 1999, 2002). ERK is phosphorylated asa result of the activation of neurotransmitter receptors in
the excitable membrane. C-fiber stimulation in peripheral
structures activates the ERK pathway in primary afferent
and dorsal horn neurons (Ji et al., 1999, 2002; Liu et al.,2004). The pERK expression was increased following an
increase in the noxious stimulus intensity in these neurons(Dai et al., 2002; Ji et al., 1999). The present study showsthat the capsaicin stimulation of the tooth pulp induced
phosphorylation of ERK in the Vi/Vc zone, Pa5 and Vc/C2zone neurons. The phosphorylation of ERK was observed
immediately after the tooth pulp stimulation (within 1 min).
It is known that the capsaicin application dominantly
activates the C-fibers in peripheral structures (Guan et al.,
2004; Lin et al., 2000). This indicates that the pERK
expression in medulla observed in the present study is
the result of the activation of the intra-pulpal C-fiber
afferents. It is clear that the capsaicin stimulation activates
primary afferent neurons, resulting in the activation of
tooth-pulp-driven neurons in Vi/Vc zone, Pa5 and Vc/C2.
Thus, an activation of intra-pulpal C-fiber afferents leads tointracellular signal transduction in tooth-pulp-driven neurons in the Vi/Vc zone and Pa5 and Vc/C2 zone via theintracellular ERK signal transduction pathway, resulting in
the inflammation-induced tooth pulp pain.

4.

Experimental proceduresThis study was approved by the Animal Experimentation Committee at Nihon University School of Dentistry, and animaltreatments were performed according to the guidelines of theInternational Association for the Study of Pain (Zimmerman,

1983). Sprague–Dawley male rats weighing between 250 and 350 g
were used.
We divided the experimental rats in 7 groups depending on thetreatment as follows: rats with capsaicin or vehicle-injected into
the first upper molar, rats with capsaicin or vehicle-injected into
the lower molar, rats with capsaicin or vehicle subcutaneously
injected into the whisker pad and naive rats without any
treatments.
4.1.

Electromyographic recording and capsaicin application

The capsaicin or vehicle-treated rats (n = 5 in each group) wereintroduced for EMG recording experiments. All procedures were
performed under sodium pentobarbital anesthesia (50 mg/kg, i.p.).
Capsaicin (Wako Co. LTD) was dissolved in 100% ethanol and 7%
Tween 80 in saline (10 mM) and injected into the upper right firstmolar pulp with the tip of paper points. The same amount ofsolvent (100% ethanol and 7% Tween 80 in saline (10 mM)) wasused as a vehicle.
Anesthesia was initially induced by inhalation of 3.0%
halothane mixed with oxygen, and a tracheal cannula wasinserted. Animals were placed on a heating pad in the dorsal
recumbent position. To allow for the application of capsaicin in
the maxillary first molar, the rats' mouth was gently opened andthe dental pulp was exposed by means of low-speed dental drill
with a round tungsten carbide bur (No. 1–4) under water cooling.
The pulp surface was covered with a small piece of cotton
soaked with physiological saline until capsaicin was applied.
After surgery, the halothane concentration was reduced (0.6–
0.8%) until noxious pressure applied to the hind-paw could
induce a weak flexion reflex of the hind limb to ensure that an
adequate level of anesthesia was maintained during theexperiment. The heart rate, percent of expired CO2 concentration and body core temperature were continuously monitored
throughout the experiment and kept within the physiologicalrange. A pair of bipolar EMG electrodes (enamel-coated stainlesssteel wire, inter-polar distance: 5 mm) was inserted into thedigastric and masseter muscles on the side ipsilateral to thechemical application. A rest period of 1–2 h was allowed after
the surgery in order to obtain a stable baseline level of EMG
activity. EMG activity in two jaw muscles was continuously
monitored before, during and after the application of capsaicin
into the molar tooth pulp. Capsaicin was applied to the tooth
pulp by a small piece of dental paper point (diameter, 0.15 mm;
length, 1.5 mm; PIERCE ABSORBENT POINTS, ISO size #15); thecavity was then quickly sealed with a small piece of cotton toprevent any possible leakage of capsaicin to the surrounding
tissues. Before the application of capsaicin, the baseline EMG
activity was monitored for 20 min, and, after the application of
the chemical, the EMG activity was continuously monitored foran additional 20 min. The EMG activity from each muscle wasamplified and rectified and integrated by the Spike 2 software(CED, Cambridge). The mean baseline level was determined by
calculating the mean EMG area over the first 20 min prior to theinjection of capsaicin into the tooth pulp. The area of theintegrated EMG activity was measured for every 1 min, andmean EMG activity was calculated.
4.2.

pERK and Fos immunohistochemistries

The procedure for capsaicin or vehicle treatment of the tooth
pulp was same as EMG recording experiment. Fifty microliters of
capsaicin or vehicle was injected into the whisker pad subcutaneously. After appropriate survival times (the survival timeafter capsaicin stimulation of the maxillary first molar: 2, 5, 10,30, 120 min, n = 5 each group; 5 min after vehicle injection into
the first upper or lower molar tooth pulp or subcutaneous
injection into the whisker pad, n = 5 each group), rats were
perfused through the aorta with 500 ml 0.9% saline followed by

BR A I N R ES E A RC H 1 0 7 2 ( 2 00 6 ) 9 9 –1 09

500 ml 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH
7.4). The naive rats were anesthetized without any surgicaltreatment and perfused with the same procedure as tooth-pulptreated rats. The whole brain including medulla and upper
cervical cord was removed and post-fixed in the same fixative
for 3 days at 4 °C. The tissues were then transferred to 20%
sucrose (w/v) in phosphate-buffered saline (PBS) for several days
for cryoprotection. Thirty-micron-thick sections were cut with afreezing microtome, and every fourth section was collected in
PBS. Free-floating tissue sections were rinsed in PBS, 10% normal
goat serum in PBS for 1 h, and then incubated in rabbit antiphospho-p44/42 MAP Kinase Antibody (1:1000, Cell Signaling
Technology) for 72 h at 4 °C. Next, the sections were incubatedin biotinylated goat anti-rabbit IgG (1:600; Vector Labs, Burlingame, CA, USA) for 2 h at room temperature. After washing, thesections were incubated in peroxidase-conjugated avidin–biotin
complex (1:100; ABC, Vector Labs) for 2 h at room temperature.
After washing in 0.05 M Tris Buffer (TB), the sections wereincubated in 0.035% 3,3′-diaminobenzidine-tetra HCl (DAB,
Sigma), 0.2% nickel ammonium sulfate and 0.05% peroxide in
0.05 M TB (pH 7.4). The sections were washed in PBS, seriallymounted on gelatin-coated slides, dehydrated in alcohols andcover slipped. For Fos immunohistochemistry, rats were perfused with same fixative used for pERK immunohistochemistry
60 min after capsaicin (n = 5) or vehicle (n = 5) injection into theupper molar tooth pulp. The medulla and the upper spinal cord
were cut, and sections were rinsed in PBS, 3% normal goat
serum (NGS) in PBS for 1 h, then incubated for 72 h with rabbit
anti-c-fos (1:20,000: c-fos ab-5, Oncogene, MA, USA) in 3% NGS at
4 °C. The following immunohistochemical procedures weresimilar to those of pERK immunohistochemistry.
The pERK- or Fos protein-LI cells were drawn under thelight microscope using camera-lucida drawing tube. The
number of pERK- or Fos protein-LI cells was counted fromevery 8th section. The total number of pERK- or Fos protein-LI
cells from three of every 8th section was calculated, and themean number of pERK-LI cells (three sections/rat) was
obtained from each animal in order to avoid the variabilityof the number of immunoreactive neurons in each section.
4.3.

Western blot

Six rats with capsaicin or vehicle injection into the upper firstmolar tooth pulp (3 rats in each group) were used for Western
blot analysis. Rats were anesthetized with sodium pentobarbital(50 mg/kg, i.p.), and upper molar tooth pulps were treated withcapsaicin. Five minutes after capsaicin injection, rats were
perfused with 500 ml of 0.1 M PB containing phosphataseinhibitor (40 mM NaF, 10 mM Na3Vo4 and 100 mM NaCl), andbrainstem and upper cervical cord were removed. The upper halfof the medulla and cervical cord were cut. Sections 1 mm rostral
to 2 mm caudal from the obex (Vi/Vc zone and Pa5) and 4 to
8 mm caudal from the obex (Vc/C2 zone) were cut from thebrainstem and upper cervical cord. Then, the lateral half of eachsection (ipsilateral and contralateral sides to capsaicin treatment) was taken for Western blot. The samples were immersed
in 200 μl of homogenization buffer (50 mM Tris–HCl, pH 7.5/5 mM
EGTA/1.4 μM β-mercaptoethanol/2 mM phenylmethylsulfonyl
fluoride/0.2 mg/ml trypsin inhibitor) and homogenized by
Dounce's homogenizer for 25 strokes. The homogenates were
centrifuged at 14,000×g for 5 min, and the supernatants weretransferred to fresh tubes. Ten micrograms of samples was
loaded for 10% sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to Immobilon-Ptransfer membrane (Millipore, Bedford, MA). The membranes
were incubated in 1% (w/v) bovine serum albumin (BSA) in PBS/
0.1% Tween 20 (BSA-PBST) for 1 h at room temperature to block
the background reaction and then incubated with polyclonal
rabbit anti-rat phospho-ERK, rabbit anti-rat nonphospho-ERK

107

antibodies for 16 h at 4 °C. After washing in PBS containing 0.1%
Tween 20 (PBST), the membranes were incubated with horseradish-peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L)(Jackson Immuno Research Laboratories, West Grove, PA)(1:10,000 dilution in BSA-PBST). The membranes were washedwith PBST for three times and then incubated with ECL reagents(Amersham, Piscataway, NJ) for 1 min. The membranes were
exposed to X-ray film (Kodak) for 2–5 min and developed.
Autoradiograms were analyzed, and the intensity of immunoreactive bands of interest was quantified by densitometric analysis
using NIH Image 1.60.
4.4.

Statistical analysis

Statistical analysis was performed using analysis of variance(ANOVA) followed by Fisher's protected least-significant difference (LSD) test, Newmann–Keuls test, Dunnett's test or Scheffetests. Student's t test or Welch's t test was also used as
appropriate. Differences were considered significant at p b 0.05.
Results are presented as means ± SD.

Acknowledgments
We thank Prof. R. Dubner for invaluable comments and Dr. D.
A. Thomas for correcting English usage on the manuscript.
This study was supported in part by Research grants from
Sato and Uemura Funds from Nihon University School of
Dentistry and Grant from Dental Research Center, Nihon
University School of Dentistry; Nihon University multidisciplinary research grant and Individual Research Grant; a Grant
from the Ministry of Education, Culture, Sports, Science andTechnology to promote multidisciplinary research projects; agrant from the Ministry of Education, Culture, Sports,
Science, and Technology to promote multidisciplinary research projects “Brain Mechanisms for Cognition, Memory
and Behavior” at Nihon University. K.R. is supported by the
NIH grant DE11964.

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