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IL-6 Induces Osteoblastic Differentiation of Periodontal Ligament Cells

¹ Departments of Hard Tissue Engineering (Periodontology) and
² Nanomedicine (DNP), Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyoku, Tokyo 113-8549, Japan; and
³ Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Japan

Correspondence: ^* corresponding author, komaki.peri{at}tmd.ac.jp

	ABSTRACT

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Interleukin (IL)-6 has been considered as an osteolytic factor involved in periodontal disease. However, the function of IL-6 in osteoblastic differentiation of periodontal ligament cells is not clear. We examined the effects of IL-6 and its soluble receptor (sIL-6R) on osteoblastic differentiation of periodontal ligament cells. Osteoblastic differentiation was induced by ascorbic acid. Osteoblast markers, including alkaline phosphatase activity and Runx2 gene expression, were examined. The mechanism of action of IL-6 on osteoblastic differentiation was evaluated by insulin-like growth factor (IGF)-I production and specific inhibitors for the IL-6-signaling molecule. IL-6/sIL-6R enhanced alkaline phosphatase activity and Runx2. Alkaline phosphatase activity was reduced by anti-IGF-I antibody. Mitogen-activated protein kinase and Janus protein tyrosine kinase inhibitors diminished alkaline phosphatase induced by IL-6/sIL-6R. We conclude that IL-6/sIL-6R increases ascorbic-acid-induced alkaline phosphatase activity through IGF-I production, implying that IL-6 acts not only as an osteolytic factor, but also as a mediator of osteoblastic differentiation in periodontal ligament cells.

Key Words: periodontal ligament • osteoblastic differentiation • IL-6 • IGF-I

	INTRODUCTION

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Periodontal disease is characterized by gingival inflammation and destruction of the periodontal attachment apparatus, leading to alveolar bone loss. Interleukin (IL)-6 is a pleiotropic cytokine that plays an important role in inflammatory reactions and also stimulates induction of osteoclast formation and bone resorption in chronic inflammatory diseases such as rheumatoid arthritis and periodontal disease (Kishimoto, 1989; Mundy, 1991; Tamura et al., 1993; Romas et al., 1996; Richards et al., 2000). IL-6 exerts its action by binding to either its membrane-bound receptor (mIL-6R) or soluble receptor (sIL-6R) and subsequent homodimerization of the signal-transducing molecule gp130, followed by activation of Janus protein tyrosine kinase (JAK), a signal transducer and activator of transcription factors (STAT)-1/3 and the mitogen-activated protein kinase (MAPK) signaling pathway (Ishihara and Hirano, 2002). Membrane-bound IL-6R is expressed in hepatocytes and subpopulations of leukocytes, but not in hematopoietic cells, osteoblasts, and fibroblasts (Bellido et al., 1997; Jones and Rose-John, 2002). Although non-IL-6R-expressing cells are not responsive to IL-6 alone, these cells can be stimulated by a complex of IL-6 and sIL-6R (IL-6/sIL-6R). This pathway has recently been termed "transsignaling" and has been reported to be involved in various important cellular functions (Jones and Rose-John, 2002).

The periodontal ligament is a unique soft tissue that connects the tooth to the alveolar bone via insertion of collagen fibers into 2 mineralized tissues—root cementum and alveolar bone proper. Periodontal ligament cells are major cellular components of the periodontal ligament. Periodontal ligament cells aligning on the dentin and alveolar bone surface contribute to the tooth-anchoring structure and are capable of forming mineralized nodules in vitro (Mukai et al., 1993; Saito et al., 2002; Yoshizawa et al., 2004). Therefore, periodontal ligament cells are considered responsible for the repair and maintenance of periodontal ligament (Bartold et al., 2000; Kalpidis and Ruben, 2002; Windisch et al., 2002; Venezia et al., 2004). Recently, the effects of IL-6 on osteoblastic differentiation have been reported (Nishimura et al., 1998; Suga et al., 2001; Malaval et al., 2005). These investigators reported that IL-6/sIL-6R induced alkaline phosphatase activity in human osteoblastic cells. Another group showed that elevated alkaline phosphatase activity was found in bone-marrow-derived mesenchymal stem cells when the cells were stimulated with IL-6/sIL-6R in the presence of dexamethasone (Erices et al., 2002).

However, the role of IL-6 in the context of osteoblastic differentiation is still controversial, and no information is available on periodontal ligament cells. We hypothesized that IL-6 plays a role not only in inflammation and periodontal bone destruction, but also in the repair and maintenance of periodontal ligament.

	MATERIALS & METHODS

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Periodontal Ligament Cells
Human periodontal ligament cells were obtained from explants of clinically healthy periodontal ligament tissues of premolar teeth taken from five individuals (ages 18–31 yrs) undergoing tooth extraction for orthodontic reasons. Informed consent was obtained from all the individuals. The experimental protocol was approved by the ethics committee of Tokyo Medical and Dental University. Periodontal ligament cells grown from periodontal ligament tissues were cultured in Dulbecco’s Modified Eagle Medium (D-MEM) (GIBCO BRL Div. of Invitrogen, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO) and 1% antibiotic-antimycotic liquid (GIBCO) in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. The cells from passages 4 to 9 were used for the experiments.

Cell Culture
We have previously reported that ascorbic acid induced osteoblastic differentiation of periodontal ligament cells, showing enhanced alkaline phosphatase activity and osteoblast-specific transcription factor Runx2 mRNA expression (Ishikawa et al., 2004; Leon et al., 2007; Mimori et al,. 2007). Therefore, we used this culture system to investigate the effects of IL-6 on osteoblastic differentiation in periodontal ligament cells. For osteoblastic differentiation, cells (5 x 10⁴ cells/mL) were seeded onto culture plates, and after 24 hrs, were cultured in D-MEM+10% FBS with 50 µg/mL of ascorbic acid (L-ascorbic acid 2-phosphate) (Sigma-Aldrich Co., St. Louis, MO, USA) in the presence or absence of recombinant human (rh) IL-6 (R&D Systems, Inc., Minneapolis, MN, USA) (50 ng/mL) and sIL-6R (R&D Systems) (40 ng/mL) for up to 7 days. For examination of insulin-like growth factor (IGF)-I function in osteoblastic differentiation, various doses of rhIGF-I (R&D Systems) (0.2–200 ng/mL) or IGF-I neutralizing antibody (Upstate Biotechnology, Inc., Lake Placid, NY, USA) (40, 80 µg/mL) were added to cell cultures in the presence of ascorbic acid and IL-6/sIL-6R for 7days. The culture medium was replaced every 3 days by D-MEM+10% FBS with indicated stimuli.

Measurement of Alkaline Phosphatase Activity
Seven days after stimulation, culture supernatants were collected for IGF-I measurement, and alkaline phosphatase activity was determined. The cells were rinsed with cold PBS and subjected to lysis in Tris-buffered saline containing 1% Triton-X100. Alkaline phosphatase activity of the lysate was assayed at 37°C for 30 min in 0.5 M Tris-HCl buffer (pH 10.5) containing 1 mM MgCl₂, with 0.5 mM para-nitrophenyl phosphate as a substrate. The absorbance was read at OD 405 nm. Alkaline phosphatase activity was expressed as the OD at 405 nm/cell number. Representative data from at least 3 experiments are shown.

Alkaline Phosphatase Staining
To examine alkaline phosphatase activity histochemically, we performed alkaline phosphatase staining as described previously (Katagiri et al., 1994). In brief, 7 days after stimulation, periodontal ligament cells were fixed with 10% formalin in PBS at room temperature for 10 min and washed 3 times with PBS. The fixed cells were incubated for 20 min at 37°C with 0.1 mg/mL of naphthol AS-MX phosphate (Sigma-Aldrich), 0.5% N,N-dimethylformamide (Sigma-Aldrich), and 0.6 mg/mL of fast blue BB salt (Sigma-Aldrich) in 0.1 M Tris-HCl, pH 8.5, and 10 mM MgCl₂. Representative data from at least 3 experiments are shown in Figs. 1 and 4.

View larger version (36K): [in this window] [in a new window]   Figure 1. IL-6/sIL-6R enhances ascorbic-acid-induced alkaline phosphatase activity in periodontal ligament cells. Periodontal ligament cells were stimulated with ascorbic acid (50 µg/mL), IL-6 (50 ng/mL), or sIL-6R (40 ng/mL) alone or in combination, as indicated. Alkaline phosphatase activity was examined by alkaline phosphatase staining (A) and alkaline phosphatase activity assay (B) (bar = 100 µm) 7 days after stimulation. *Significantly different from control (p < 0.05). **Significantly different from ascorbic acid (p < 0.001). Data in graphs are presented as the mean ± SD of 3 experiments.

View larger version (69K): [in this window] [in a new window]   Figure 4. Effects of MAPK and JAK/STAT inhibitors on alkaline phosphatase activity. PD98059 was used to inhibit ERK, and AG49 and JAK inhibitor I were used for JAK/STAT signaling inhibition. Periodontal ligament cells were pre-incubated with DMSO, PD98059 (20 µM), AG490 (AG, 20 µM), or JAK inhibitor I (JAK, 15 nM) for 30 min and cultured with or without ascorbic acid, IL-6, and sIL-6R for 7 days. We examined alkaline phosphatase staining (A,C) and alkaline phosphatase activity (B,D) (bar = 100 µm) to identify the effects of inhibitors of alkaline phosphatase activity on periodontal ligament cells. *Significantly different from ascorbic acid+IL-6/sIL-6R (p < 0.05). **Significantly different from ascorbic acid+IL-6/sIL-6R (p < 0.05). Data in graphs are presented as the mean ± SD of 3 experiments.

Signaling Pathways
It is known that the formation of IL-6 and the IL-6 receptor complex triggers the activation of a JAK/STAT and MAPK cascade. To examine the signaling pathways involved in alkaline phosphatase induction in periodontal ligament cells, we investigated the effects of the MAPK inhibitor, PD98059, and JAK inhibitors, AG490 and JAK inhibitor I, on ascorbic acid+IL-6/sIL-6R-induced alkaline phosphatase activity. Periodontal ligament cells were pre-incubated for 30 min with DMSO, PD98059 (Sigma-Aldrich) (20 µM), JAK inhibitor 1 (Calbiochem, La Jolla, CA, USA) (15 nM), or AG480 (Calbiochem) (20 µM) prior to stimulation with ascorbic acid and IL-6/sIL-6R. The medium containing inhibitor, ascorbic acid, IL-6, and sIL-6R was changed every 3 days for 7days, followed by the assessment of alkaline phosphatase activity. Representative data from at least 3 experiments are shown.

Real-time Polymerase Chain-reaction (Real-time PCR)
Total RNA was prepared with an ISOGEN kit (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. A 2-µg quantity of total RNA was used for cDNA synthesis with Ready-To-Go You-Prime First-Strand Beads (Amersham Biosciences Corp., Piscataway, NJ, USA) according to the manufacturer’s instructions. Quantitative real-time PCR was carried out with LightCycler FastStart DNA Master SYBR Green I, Light Cycler Primer for human GAPDH, and Runx2 (Roche Diagnostic KK, Tokyo, Japan) according to the manufacturer’s protocol. Control DNA of the primer set was used as a positive control, and PCR-grade water was used as a negative control. Amplification conditions were 95°C for 10 min for denaturation, 35 cycles at 95°C for 10 sec, 68°C for 10 sec, and 72°C for 16 sec, followed by a melting curve from 58°C to 95°C. The reaction product was quantified with the LightCycler Software Ver. 3.5 (Roche Diagnostic KK). The value was normalized to an internal control (GAPDH). Representative data from at least 3 experiments are shown.

Statistical Analysis
Data are expressed as mean ± standard deviation (SD). Statistical significance was determined by analysis of variance with StatView 4.0 for the Macintosh (SAS Institute, Cary, NC, USA). Significant differences between the different groups were determined by two-way ANOVA with Fisher’s post hoc comparisons. A probability of P < 0.05 was considered to be significant.

	RESULTS

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Effects of IL-6 and sIL-6R on Osteoblastic Differentiation
The addition of IL-6/sIL-6R enhanced alkaline phosphatase activity, and the alkaline phosphatase activity was synergistically increased (p < 0.001) when periodontal ligament cells were incubated with ascorbic acid and IL-6/sIL-6R (Figs. 1A, 1B). Ascorbic acid+IL-6/sIL-6R treatment led to an increased number of alkaline-phosphatase-positive cells and a higher intensity of alkaline phosphatase staining compared with other experimental treatments.

IL-6/sIL-6R Stimulates Runx2 Gene Expression
Next, we investigated whether IL-6/sIL-6R could induce osteoblast-specific transcription factor Runx2 gene expression in periodontal ligament cells (Fig. 2). When the cells were stimulated with either IL-6 or sIL-6R alone, Runx2 expression was not changed. Ascorbic acid increased Runx2 expression. In the presence of ascorbic acid, IL-6 or sIL-6R slightly enhanced Runx2 expression on day 6. IL-6/sIL-6R dramatically increased ascorbic-acid-induced-Runx2 expression at both days 3 and 6.

View larger version (23K): [in this window] [in a new window]   Figure 2. Effects of ascorbic acid, IL-6, and sIL-6R on Runx2 gene expression. Periodontal ligament cells were stimulated with ascorbic acid (50 µg/mL), IL-6 (50 ng/mL), or sIL-6R (40 ng/mL) alone or in combination, as indicated. Runx2 gene expression was examined by real-time PCR with cDNA from periodontal ligament cells at 3 and 6 days after stimulation. The expression levels of transcripts were compared with the level of an internal control (GAPDH). Three independent experiments were performed, and representative results are shown.

View larger version (15K): [in this window] [in a new window]   Figure 3. Involvement of IGF-I in IL-6/sIL-6R-induced alkaline phosphatase activity. Periodontal ligament cells were stimulated with indicated combinations of IL-6 (50 ng/mL), sIL-6R (40 ng/mL), ascorbic acid (50 µg/mL), IGF-I (0.2–200 ng/mL), or anti-IGF-I antibody (40 or 80 µg/mL) for 7 days. Production of IGF-I was examined by enzyme-linked immunosorbent assay (A). Effect of exogenous IGF-I on alkaline phosphatase activity in periodontal ligament cells was investigated (B). Effect of IGF-I antibody on ascorbic acid+IL-6/sIL-6R-induced alkaline phosphatase activity in periodontal ligament cells was studied (C). *Significantly different from control (p < 0.05). **Significantly different from ascorbic acid (p < 0.001). ***Significantly different from ascorbic acid+IL-6/sIL-6R (p < 0.001). Data in graphs are presented as the mean ± SD of 3 experiments.

	DISCUSSION

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We showed that IL-6/sIL-6R synergistically enhanced alkaline phosphatase activity and Runx2 expression in ascorbic-acid-challenged periodontal ligament cells, suggesting that ascorbic acid and IL-6/sIL-6R induce osteoblastic differentiation of periodontal ligament cells. We have reported that ascorbic acid induces osteoblastic differentiation of periodontal ligament cells through production of type-I collagen and expression of 1β2 integrin (Ishikawa et al., 2004). However, IL-6/sIL-6R did not change type-I collagen production and β1 integrin in periodontal ligament cells (data not shown). Analysis of these data suggests that IL-6/sIL-6R has another mechanism for enhancing ascorbic-acid-induced alkaline phosphatase activity. In this study, IL-6/sIL-6R significantly induced IGF-I production in periodontal ligament cells, which is consistent with a previous report in osteoblastic cells (Franchimont et al., 1997), and neutralizing antibody to IGF-I inhibited ascorbic acid+IL-6/sIL-6R-induced alkaline phosphatase activity, strongly suggesting that IGF-I mediated alkaline phosphatase activity when periodontal ligament cells were stimulated with ascorbic acid+IL-6/sIL-6R. However, because IGF-I neutralizing antibody did not inhibit alkaline phosphatase activity to control levels (approximately 70% of the ascorbic acid+IL-6/sIL-6R group), it is possible that another mechanism still remains unclear. It has been reported that IL-6/sIL-6R enhanced BMP6 expression in bone-marrow-derived mesenchymal stem cells (Yeh et al., 2002). Although we did not clarify the involvement of BMP6 in our study, the production of local factors, including growth factors, may mediate osteoblastic differentiation of periodontal ligament cells.

Gp130 is found in almost all organs and cells, whereas mIL-6R expression differs among cell types, suggesting different responses to IL-6, depending on the expression pattern of mIL-6R (Saito et al., 1992). We found high levels of gp130 expression, whereas the expression of mIL-6R was weak in periodontal ligament cells (data not shown). We showed that IL-6 alone failed to change the levels of both basal- and ascorbic-acid-induced alkaline phosphatase activity in periodontal ligament cells. Analysis of these data suggests that mIL-6R is not sufficient to transduce IL-6 signals. Thus, the transsignaling by IL-6/sIL-6R appears to have an important role in the regulation of IL-6 function on periodontal ligament cells, as in other non-mIL-6R-expressing cells, including osteoblasts and fibroblasts (Bellido et al., 1997). It is known that IL-6/sIL-6R induces STAT1/3 activation through JAK1 and JAK3 and also stimulates the MAPK-dependent signaling pathway (Nakajima et al., 1996; Heinrich et al., 1998). It has been demonstrated that ascorbic acid induces collagen synthesis, which in turn binds to 1β2 integrin and activates MAPK in osteoblast cell line cells (Xiao et al., 1997, 2000). From these previous reports, we conclude that it is possible that STAT1/3 and MAPK signaling mediates alkaline phosphatase activity in periodontal ligament cells stimulated with a combination of ascorbic acid and IL-6/sIL-6R. We showed that PD98059, AG490, and JAK inhibitor I down-regulated ascorbic acid+IL-6/sIL-6R-enhanced alkaline phosphatase activity in periodontal ligament cells. These results suggest the involvement of the MAPK and JAK pathways in osteoblastic differentiation of periodontal ligament cells. Furthermore, analysis of our data suggested that IGF-I secreted after IL-6/sIL-6R and ascorbic acid treatment had an important role in the differentiation of periodontal ligament cells. Previously, it was reported that IGF-I stimulates MAPK in many cell types, including osteoblasts, and that IGF-I induced osterix expression, one of the most crucial transcription factors in osteoblastic differentiation, through MAPK components, including ERK, p38, and JNK (Celil and Campbell, 2005). In this study, we did not examine the activation of MAPK and JAK/STAT signaling by IGF-I in periodontal ligament cells. Further study is needed to investigate this point.

Recently, IL-6 has been demonstrated to induce osteoblastic differentiation of mesenchymal stem cells (Franchimont et al., 2005). Some have suggested a functional role of IL-6 in bone formation with a STAT-1/3 and MAPK transgenic mice model (Sims et al., 2004). These investigators demonstrated higher turnover of bone in STAT-1/3 transgenic mice, and showed that osteoblast differentiation was significantly inhibited in IL-6 null mice with the MAPK transgenic phenotype, suggesting that IL-6 has a key role in the formation of osteoblasts, but not of osteoclasts. Furthermore, periodontal ligament cells have type I collagen predominantly in their extracellular matrix and a higher turnover of type I collagen, which implies the functional importance of type I collagen in periodontal ligament cells (Yamada et al., 2001). It is thus possible that IL-6/sIL-6R enhances osteoblastic differentiation of periodontal ligament cells in periodontal lesions. It has been reported that IL-11, one of the IL-6 family of cytokines, significantly inhibited bone destruction in ligature-induced periodontal disease in dogs, suggesting an anti-destructive function of gp130 cytokine in periodontal disease (Martuscelli et al., 2000). However, it is still unclear what mechanism controls the ostoblastic and pro-inflammatory effects of IL-6 in periodontal disease. Further investigation is needed to clarify this question.

In conclusion, we demonstrated that IL-6/sIL-6R enhanced ascorbic-acid-induced alkaline phosphatase activity by induction of IGF-I production in periodontal ligament cells. This result suggests that IL-6 plays a role not only in inflammation, but also in remodeling of alveolar bone to maintain periodontal tissue by regulating osteoblastic differentiation of periodontal ligament cells.

	ACKNOWLEDGMENTS

 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to KI and MK.

Received for publication December 18, 2006. Revision received February 19, 2008. Accepted for publication July 10, 2008.

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Journal of Dental Research, Vol. 87, No. 10, 937-942 (2008)
DOI: 10.1177/154405910808701002


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