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Expression and effect of tumor necrosis factor-α in human dental
follicle cells in vitro
Yingchun Bi, DDS, PhD, Zhu Lin, DDS, PhD, Zuolin Jin, DDS, PhD, Hong Qian DDS,
PhD, Xuepeng Chen, DDS, PhD, Yongkuan Zhang, PhD, and Xueying Han, DDS
Department of Orthodontics, College of Stomatology, The Fourth Military Medical University, Xi’an,
P. R. China
Purpose: To investigate (1) the effect of interleukin-1α (IL-1α) on tumor necrosis factor-α (TNF-α) expression,(2) the induced effect of TNF-α on the expression of monocyte chemoattractant protein-1 (MCP-1), and (3) the
corresponding signal transduction pathway in the human dental follicle cells (HDFCs).
Materials and Methods: Primary HDFCs were cultured. The fifth passaged HDFCs were incubated with IL-1α.
Then examined the expression of TNF-α by reverse transcription polymerase chain reaction (RT-PCR) andenzyme-linked immunosorbent assays (ELISA). The expression of MCP-1 was examined after HDFCs
incubatied with TNF-α by ELISA and RT-PCR. Furthermore, the induction of MCP-1 mRNA expression by
TNF-α was inhibited by SB203580, SP600125, and PD98059.
Results: The results of RT-PCR and ELISA revealed that IL-1α increases the TNF-α gene expression andsecretion significantly at concentration of 25-100 ng/mL (p<0.05). Furthermore, TNF-α enhances MCP-1 gene
expression and secretion at concentration of 10-100 ng/mL (p<0.05) and the action is inhibited by SP600125, thespecial inhibitor of c-Jun N-terminal kinase (JNK).
Conclusion: IL-1α induces expression of TNF-α by HDFCs. One of the actions of TNF-α in tooth eruption
may be that it enhances MCP-1 gene expression and secretion and these events require JNK activity, which
recruits monocytes into the dental follicle, where these cells are fused to form osteoclasts to resorb alveolar bone
for the formation of an eruption pathway. (Int Chin J Dent 2007; 7: 35-42.)
Key Words: dental follicle, interleukin-1α, monocyte chemoattractant protein-1, tumor necrosis factor-α.

Introduction
A critical cellular event of tooth eruption is the influx of mononuclear cells into the dental follicle, the loose
ectomesenchyme derived connective tissue surrounding the enamel organ and the dental papilla of thedeveloping tooth germ prior to eruption. The mononuclear cells recruited into dental follicle are fused to form
osteoclasts needed for bone resorption.1 Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that
often is overexpressed in a number of disease states such as sepsis syndrome, rheumatoid arthritis, inflammatory
bowel disease,2 and periodontitis.3 TNF-α induces osteoclast differentiation and bone resorption by stimulating
osteoclast precursors. It gains importance as a local regulator of bone cell function for good reason. TNF-αtreatment of cultured bone explants or cell cultures of mineralizing osteoblasts caused increases calcium releaseand suppression of matrix protein production, suggesting stimulation of bone resorption and inhibition offormation.4-6 In addition, increased production of TNF-α is demonstrated as part of the overall cytokine stimulus,
and blockade of TNF-α action is shown to alleviate bone loss.7-8 It was reported that TNF-α is maximally
expressed in dental follicle of postnatal day 9 rats regulated by interleukin-1α (IL-1α) and TNF-α stimulates theexpression of potential tooth eruption genes monocyte chemoattractant protein-1 (MCP-1) and vascularendothelial growth factor (VEGF). There is a second minor burst of osteoclasts seen on the alveolar bonesurrounding the first molar and we have proposed that the maximum expression of TNF-α at day 9 may be
involved to up-regulate the second round of osteoclastogenesis.9
Recent studies have shown no single mechanism is responsible for TNF-α action on bone cell; activation of acomplex signal pathway drives gene transcription in a direction that produces a skeletal catabolic state. Kumar

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et al. demonstrated that in osteoblasts significant activation of p38 mitogen-activated protein kinase (MAPK)
was observed following treatment with TNF-α while inhibitors of p38 MAPK have been shown to reduce TNF
stimulation of osteoclastogenesis.10 Using a human peripheral blood mononuclear cells (PBMC) culture model,
HB Kwak et al. reported that TNF-α regulates osteoclast differentiation through p21WAF1/Cip1 expression andfurther shows that these events require c-Jun N-terminal kinase (JNK) activity.11 However, even the regulationof TNF-α has been studied in different cell types, no studies to date have addressed intracellular signaling andmolecular regulation mechanisms on human dental follicle cells (HDFCs).
In this study, we investigated whether TNF-α is normally expressed in the dental follicle cells, and the levelsof the effect of TNF-α itself on the expression of genes in HDFCs that may relate to tooth eruption. In addition,
signal transduction pathways that TNF-α regulates in HDFCs were also investigated using specific inhibitors.

Materials and Methods
Culture of HDFCs
HDFCs were established and cultured as previously described.12 Prior to commencement, the study protocol
was approved by Ethics Committee of the Fourth Military Medical University. Normal adolescents impacted
third molars were surgically removed and collected which needed for orthodontics treatment after informed
consent. The age of the patients varied between 11 and 15 years. Attached dental follicles were separated fromthe mineralized tooth. The surfaces of the follicle tissues were cleaned and minced by using a sterilized scalpel.
Also dental papilla tissue was discarded. Tissues were digested in a solution of 0.1 U/mL collagenase type I and1 U/mL dispase (Roche, Mannheim, Germany) for 1 hour at 37˚C. Minced and digested tissues of dental follicle
explants were seeded into 60 mm plates with 25 mL Dulbecco's modified Eagle medium (DMEM, HyClone,
Logan, UT, USA) containing 10% fetal bovine serum (FBS) and 1 mmol/L sodium pyruvate, in an incubator at
37˚C in an atmosphere of 5% CO2. After single cells had been attached to the plastic surface, non-adherent cells
were removed by change of medium. Plastic adherent cells were continuously propagated until confluence.
HDFCs of passage 5 were grown in 250 mL T-flasks until confluence.

Prior to treatments, cells wereserum-deprived DMEM containing 0.2% fatty-acid-free and growth-factor-depleted bovine serum albumin(BSA) for 12 hours.
Incubation of HDFCs with IL-1α
The cells were incubated in serum-free minimal essential medium containing recombinant human IL-1α(PeproTech Rock Hill, NJ, USA) for the designated concentration-dependent studies. The cells were incubatedwith IL-1α 0, 5, 10, 25, 50, and 100 ng/mL9 for 6 hours. After the treatments, the cells were collected for RNA
isolation and cell culture supernates were collected for TNF-α level measured by enzyme-linked immunosorbent
assays (ELISA). Each experiment was repeated at least three times. Controls were incubated in serum-free
DMEM containing BSA only.
Isolation of total RNA
Total cellular RNA was isolated from each culture using an RNeasy Mini Kit (Qiagen, Chatsworth, CA, USA),according to protocols provided by the manufacturer. To remove contaminating genomic DNA, the RNA
samples were treated with RNase-free DNase I (Qiagen) at 37°C for 30 minutes.
Reverse transcription polymerase chain reaction
First, 2 µg of total RNA was used as a template to synthesize first-strand complementary DNA (cDNA) with36

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Int Chin J Dent 2007; 7: 35-42.

oligo (dT) primer and reverse transcriptase, using the Omniscript RT Kit (Qiagen). Then 1 µL of the cDNA
mixture was subjected to polymerase chain reaction (PCR) amplification using specific primers. The specificprimers for the genes of TNF-α, MCP-1 and β-actin were designed using Primer Premier 5 (Premier Biosoft Ltd.,
Palo Alto, CA, USA). Primers were synthesized by BioAsia Corp. (Shanghai, China). The primer sequencesused in this study are shown in Table 1.
Table 1.

Primer sequences used in semi-quantitative RT-PCR.
Forward

ReverseTNF-α (product 491 bp) 5′-GAAATGGAGGCAATAGGTT-3′
MCP-1 (product 308 bp) 5′-CTTCTGTGCCTGCTGCTC-3′
bactin (product 326 bp) 5′-CCGGATCCATCTCTTGCTCGAAGTCCA-3′5′-AGCCGTGGGTCAGTATGT-3′5′-AGATTCTTGGGTTGTGGA-3′5′-AGAGGCATACAGGGACAACACA-3′

A total of 40 cycles of PCR was performed by mixing 3 µL cDNA with dNTP, primers, PCR buffer, and Taq
DNA polymerase (Applied Biosystems, Foster City, CA, USA) to 25 µL in each reaction with the amplification
protocol of 94˚C for 30 s (denatureation), 58.5˚C for 2 s (annealing), and 74˚C for 30 s (extention). The
amplification products were electrophoresed on 2% agarose gels (Amresco, Solon, OH, USA) and visualized byethidium bromide staining. The relative intensities of the gel bands were measured using NIH Image software,and the results were normalized to the mRNA level of actin, a housekeeping enzyme. We performed theseexperiments using samples from at least three different cell preparations, and quantification of mRNA wasconfirmed using the same cell sample at least in triplicate.
ELISA for the quantitative determination of TNF-α/MCP-1 concentration
In order to quantify the concentration of TNF-α secreted after incubating the dental follicle cells with TNF-α
and that of MCP-1 secreted after incubating the dental follicle cells with TNF-α, ELISA kits (R&D Systems,
Minneapolis, MN, USA) were employed using a monoclonal antibody for human TNF-α and MCP-1 that had
been precoated onto micro-plates. Measurements were performed in triplicate on samples diluted 1:10 according
to the manufacturer’s instructions. In brief, 100 µL assay buffer, 50 µL sample, and 50 µL detection antibody
were added to each well. After incubation for 16 hours at 4°C, the wells were washed with wash buffer, then
200 µL streptavidin-horseradish peroxidase conjugate was added to each well followed by incubation for 60
minutes at room temperature. After further washing, substrate was added to all wells and incubated for 20
minutes, followed by stop solution. The absorption was determined with a micro-plate reader model 550(Bio-Rad, Hercules, CA, USA) at 450 nm against 570 nm as a reference. The intensity of the blue color
measured was in proportion to the amount of human TNF-α and MCP-1 bound. All experiments were repeatedthree times.
Inhibitors
Signal transduction pathways that mediated cellular responses to TNF-α were investigated using specificinhibitors: SB203580 (25 µmol/L) to inhibit p38MARK, SP600125 (15 µmol/L) to inhibit JNK, and PD98059(50 µmol/L) for specific inhibition of a extracellular signal-regulated kinase (ERK). All inhibitors were obtained
from Sigma (St. Louis, MO, USA) and the concentrations used were the effective doses previously reported.13
After the HDFCs had been pre-incubated in the presence of each inhibitor for 30 minutes to permit thesecompounds to penetrate the cells and block their respective pathways, 10 ng/mL TNF-α was applied in culture37

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Int Chin J Dent 2007; 7: 35-42.

for 6 hours. Total RNA was extracted from the cells, and the expression levels of MCP-1 mRNA were
determined by RT-PCR.
Statistical analysis
Mean values with standard deviations (SD) were calculated.

Some data were subjected to multiple

measurement analysis of variances (ANOVA), and Student’s t test was used to analyze differences between thecultures tested. A p-value of less than 0.05 was accepted as significant.

Results
Effect of IL-1α on expression of TNF-α mRNA and protein in HDFCs IL-1α
We first determined the TNF-α mRNA espression in HDFCs in response to IL-1α Cells were treated with
IL-1α, the cell were harvested, and the level of expression mRNA was determined by RT-PCR. The expressionof TNF-α mRNA was increased significantly by IL-1α with concentration of 25, 50, and 100 ng/mL in
comparison with the control (IL-1α, 0 ng/mL) cultures (Fig. 1A). When the level of TNF-α protein in the
induced cultures was analyzed by ELISA, the increase of TNF-α protein (Fig. 1B) correlated with theaccumulation in TNF-α mRNA expression.

Fig. 1. Effect of IL-1α on expression of TNF-α mRNA and protein in HDFCs.
In a concentration-dependent manner, cells were stimulated for 6 hours in the IL-1α concentration indicated (A and B). Cells
were collected for examination of TNF-α mRNA by RT-PCR and supernatants were analyzed for TNF-α concentration by ELISA.
Mean values with SDs of three independent experiments are presented. *Statistically significant difference is shown above
compared to control group (IL-1α, 0 ng/mL), p<0.05.

Effect of TNF-α on expression of MCP-1 mRNA and protein in HDFCs
Fig. 2A shows how different concentration of TNF-α affect the MCP-1 mRNA expression level in HDFCs.
TNF-α concentration at 10, 25, 50, or 100 ng/mL caused a significant increase in MCP-1 mRNA expression
compared to that in control (0 ng/mL) cultures (Fig. 2A). These results were confirmed by ELISA for MCP-1
protein in the conditioned medium from these cells (Fig. 2B).

Fig. 2. Effect of TNF-α induced expression of MCP-1 protein (B), mRNA (A) in HDFCs.
The cells were stimulated for 6 hours in the TNF-α concentration indicated (A and B). Total RNAs were extracted, and the levels
of mRNA were analyzed by RT-PCR (A). MCP-1 protein levels in the supernatants were measured by ELISA (B). Mean values
with SDs of three independent experiments are presented. *Statistically significant difference is shown above compared tocontrol group (TNF-α, 0 ng/mL), p<0.05.

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Effects of inhibitors on induction of MCP-1 mRNA expression by TNF-α in HDFCs
The mechanisms of the translating signal induced by TNF-α which activates increased MCP-1 gene
expression are as yet unidentified. To investigate whether the up-regulation of MCP-1 mRNA by TNF-α wasdependent on de novo protein synthesis, we treated the cells with aforementioned special inhibitor in Materials
and Methods. The effect was inhibited by SP600125 but not by SB203580, or PD98059, suggesting that it
depended on JNK pathway (Fig. 3).

Fig. 3. Effects of inhibitors on induction of MCP-1 mRNA expression in TNF-α stimulated HDFCs.
The cells were pretreated with SB203580 (25 µmol/L), SP600125 (15 µmol/L), and PD98059 (50 µmol/L) 30 minutes,
post-treated with or without TNF-α (10 ng/mL) 6 hours. Total RNAs were extracted from the cells, and expression levels of
MCP-1 mRNA were determined by RT-PCR as described in Materials and Methods. (A) Agarose gel electrophoresis of the
products of PCR using specific primers for MCP-1 or β-actin. (B) Relative density of the MCP-1 mRNA to β-actin mRNA. Meanvalues with SDs of three independent experiments are presented. *Statistically significant difference is shown above compared#
to control group (TNF-α, 0 ng/mL), p<0.05. Statistically significant difference is shown above compared to TNF-α (10 ng/mL)group.

Discussion
Tooth eruption is a localized event and a cascade of molecular signals generated in the dental follicle andstellate reticulum appears to initiate its onset. Consequently, mononuclear cells are recruited into the follicle and,in turn, fuse to become osteoclasts needed to resorb the alveolar bone to form an eruption pathway. IL-1α ispresent early postnatally in the stellate reticulum of the first mandibular molars.14 In tooth eruption, IL-1α may
play an important role that injection of IL-α accelerated eruption by two days in rats.15 In knockout mice lacking
a functional type I receptor for interleukin-1α (IL-1R) tooth eruption is delayed for two days in molars and one
day in incisors.16 Recent researches show that IL-1α enhances CSF-1,17,18 MCP-1,19 and NF-κB20 expression
which are responsible for tooth eruption. In addition, Kawashima et al. reported that IL-1α plays a crucial partin periapical bone resorption and high correlation with TNF-α, which seems to relate to their co-stimulatory
effects.21
TNF-α is one of well recognized members of the TNF family that play an important physiological and/or
pathological role in bone metabolism. The predominant action of TNF-α in osteoclast formation and function is
likely mediated by directly targeting osteoclast precursors and mature osteoclasts, i.e., the binding of TNF-α toits receptors on osteoclast precursors/osteoclasts initiates various signaling cascades shown in, leading to themodulation of osteoclast formation, function and survival.22,23 TNF-α is expressed slightly in the dental follicleof the first mandibular molar of the rat beginning at day 3 postnatally, but maximal expression is seen at day 9, atime that correlates with a slight burst of osteoclast formation seen at day 10 postnatally.9 In our study, HDFCs
in vitro does not express constitutively the TNF-α but does express it after incubation with IL-1α. There is a
maximal expression of TNF-α at 25 ng/mL IL-1α. The expression of TNF-α decreased at 50-100 ng/mL IL-1α.

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But statistically significant difference is shown above compared to control group (IL-1α, 0 ng/mL). The result issimilar to that of Wise.9 Although the chronology of the localization of the potential eruption molecules are notclear during human tooth eruption, we speculate that IL-1α in the stellate reticulum enhances the expression of
TNF-α in the dental follicle which participates in the formation of osteoclasts. Thus paracrine signalling may
occur, whereby molecules in the stellate reticulum affect the cells in the adjacent dental follicle (or vice versa) toinitiate eruption.
MCP-1 is a member of the chemokine family of cytokines. MCP-1, a single polypeptide chain (148 aa) with asignal peptide domain (23 aa residues), is present and is secreted as mature MCP-1 (125 aa) in several cells suchas osteoblasts,24 mesangial cells,25 endothelial cells and fibroblasts,26 alveolar macrophages,27 and gingival
tissues.28 The fact that fibroblasts express the MCP-1 gene is particularly interesting because the dental follicle
is primarily fibroblastic in nature. The principal function of MCP-1 is thought to be the stimulation of monocyte
recruitment. Que et al. reported that in vivo MCP-1 is expressed to recruit the mononuclear cells to the dental
follicle to initiate eruption, with maximal gene expression at day 3 in the rat.29 However, comparing MCP-1−/−
mice with wild-type controls during tooth eruption Graves et al demonstrated that at early time points, monocyte
recruitment occurs by MCP-1 independent mechanisms while at a later time point, MCP-1 may play acontributory role in the recruitment of monocytic cells.30 Many of the eruption molecules, such as IL-1α,
TGF-β1,31 parathyroid hormone-related protein (PTHrP),32 TNF-α,9 in turn, have been shown to enhance thegene expression of MCP-1. In our experiment there is a maximal increase in expression of MCP-1 at 10 ng/mL
concentration of TNF-α, followed by a progressive decrease at higher concentration. This study shows that
TNF-α can enhance the expression of the MCP-1 gene to bring about the cellular events of eruption -recruitmentof monocytes into the dental follicle, their fusion to form osteoclasts and bone resorption by these osteoclasts toform an eruption pathway.
A major mechanism through which signals are transmitted from environmental stimuli to the nucleus involves
activation of kinase related to the mitogen-activated protein (MAP) kinase superfamily. To date, at least threesubgroups of MAP kinase family members have been identified to be involved in a wide range of cellularresponses to extracellular signals. They are extracellular signal-regulated kinases (ERKs), JNKs, and p38
MAPK. These activated kinases initiate a cascade of protein phosphorylation that activates nuclear transcription
factors, such as NF-κB, and AP-1. These transcription factors subsequently promote expression of inflammatory
cytokines, chemokines. Our studies are based on the observation that TNF-α causes p38, JNK, and ERK
activation. In our study, inhibition of MCP-1 mRNA expression by TNF-α is abrogated by pretreatment withSP600125 (Fig. 3). This experiment indicates that the JNK-MAPK pathway is involved in MCP-1 release.
Westra et al.33 reported that TNF-α is the major inducer of endothelial cell activation and is the stimulator of
inflammatory signal transduction pathway involving p38 MAPK but the p38 MAPK inhibitor significant reducesmRNA expression and protein production of IL-6, IL-8 and MCP-1, which shows that TNF-α induces MCP-1
by p38 MAPK signaling pathways in endothelial cells. However, Hanazawa et al.34 reported that the biologicalresponses of MCP-1 synthesis in osteoblasts MC3T3-El cells induced by TNF-α are regulated via c-fos pathway.
We obtain the same results as that Chen et al.35 reported that both the p42/44 MAPK and the JNK pathways
coordinately modulate TNF-α induced CCL2/MCP-1 expression in rat vascular smooth muscle cells (VSMCs).
However, in other rat cell types or in human cell systems, p38 MAPK was reported to mediate the induction of
MCP-1 elicited by TNF-α.36-38 Thus, the MAPK signals mediating TNF-α dependent MCP-1 expression may

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vary by cell types and species.
We finally conclude that IL-1α induces an increase of TNF-α synthesis and mRNA expression in cultured
HDFCs. TNF-α enhances MCP-1 synthesis and mRNA expression. Furthermore, we have evidenced that
SP600125, the special inhibitor of JNK, suppresses the TNF-α induced MCP-1 mRNA expression. Thesefindings may be important for understanding the effect of TNF-α in tooth eruption.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No. 30400510) to Zuolin Jin.

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Correspondence to:Dr. Zuolin Jin
Department of Orthodontics, College of Stomatology, The Fourth Military Medical University
145 Changle Xi Road, Xi’an, 710032, P. R. China
Fax: +86-29-83223047 E-mail: zuolinj@fmmu.edu.cn
Received January 30, 2007. Accepted April 17, 2007.
Copyright ©2007 by the International Chinese Journal of Dentistry.

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