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Mechanism of Cyclosporine-induced Overgrowth in Gingiva
H.-J. Chae2,
M.-S. Ha1,
D.-H. Yun1,
H.-O. Pae3,
H.-T. Chung3,
S.-W. Chae2,
Y.-K. Jung4 and
H.-R. Kim1,*
1 Department of Dental Pharmacology and Wonkwang Biomaterial Implant Research Institute, School of Dentistry, Wonkwang University, Iksan, Chonbuk, South Korea;
2 Department of Pharmacology and Institute of Cardiovascular Research, Medical School, Chonbuk National University, Jeonju, Chonbuk, South Korea;
3 Department of Microbiology and Immunology, School of Medicine, Wonkwang University, Iksan, Chonbuk, South Korea; and
4 Department of Biological Science, Seoul National University, Seoul, South Korea
Correspondence: * corresponding author, hrkimdp{at}wonkwang.ac.kr
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ABSTRACT
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Cyclosporine A (CsA) is a widely used immunosuppressant but with significant side-effects, such as gingival overgrowth. This study investigates how CsA induces gingival proliferation and shows the effects of the CsA-associated signaling messengers, IL-6 and TGF-β1, on gingival proliferation. CsA increased both IL-6 and TGF-β1 levels. In addition to CsA, an IL-6 or TGF-β1 treatment also induced gingival fibroblast proliferation. Inhibiting the cytokine resulted in the suppression of CsA-induced proliferation. MAPKs and PI3K are known to be involved in cell proliferation. Therefore, the effect of CsA on the kinase activities was examined. The results showed that both p38 MAPK and PI3K are essential for gingival fibroblast proliferation. TGF-β1 and IL-6 and their associated signaling transduction may be novel bona fide molecular targets for the prevention of gingival overgrowth in CsA-treated patients. (Abbreviations: MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase.)
Key Words: Cyclosporine A • human gingival fibroblast
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INTRODUCTION
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Generalized gingival overgrowth is the result of several factors, including inflammation, leukemia, inheritance, and drugs. The most frequent form is caused by drugs such as phenytoin, nifedipine, and cyclosporine A (CsA). Gingival overgrowth as a clinical outcome presents as an increased gingival volume, including an increased number of cells and a higher level of extracellular matrix production. Despite extensive research, the mechanism leading to the accumulation of abnormal amounts of gingival tissue in CsA-induced gingival overgrowth is unclear. Fibroblasts are the main cell type residing in the gingival connective tissue, and are responsible for the formation and turnover of the extracellular matrix. Studies on the effect of CsA on gingival fibroblast activity have reported conflicting findings, and it is uncertain if CsA can induce gingival overgrowth by directly altering the function of fibroblasts (Harkonen, 1999; Voulgari and Drosos, 2002).
Two cytokines, interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β1), play regulatory roles in the turnover of the periodontal tissue. The dramatic increase in IL-6 expression in the cells within the gingival connective tissue has been reported to be a histological feature of CsA-induced gingival lesions (Myrillas et al., 1999). This report is consistent with the findings of increased IL-6 levels within renal allografts and in the serum and urine of CsA-treated patients. IL-6 appears to target connective tissue cells, such as fibroblasts, by both enhancing their proliferation and regulating collagen and glycosaminoglycan synthesis (Wahl, 1985).
Recent studies have showed that CsA regulates the transcription of TGF-β1 (Cotrim et al., 2003; Ruhl et al., 2004). This suggests that TGF-β1 assists in decreasing the proteolytic activity of human gingival fibroblasts in CsA-induced gingival overgrowth in an autocrine manner. Furthermore, this cytokine plays a pathogenic role in fibrotic diseases such as pulmonary and gingival fibroses (Nakao et al., 1995). Previous studies support the theory that CsA increases the level of TGF-β1 in gingival crevicular fluid (GCF) samples from CsA-treated patients (Buduneli et al., 2001). However, it has been suggested that the TGF-β1 level is not the only factor responsible for the CsA-induced gingival overgrowth observed, because the cytokine levels of the groups exhibiting gingival overgrowth were similar to those of sites that did not (Buduneli et al,. 2001). Complex interactions between various mediators of inflammation and tissue-modeling might be involved in the pathogenic mechanisms of this side-effect.
This study examined the proliferation of gingival fibroblasts, which is an axis of gingival overgrowth, after exposing them to CsA. It is possible that some inflammatory cytokines, such as IL-6 or TGF-β1, can play an important role in CsA-induced gingival proliferation. Therefore, this study examined whether CsA releases IL-6 or TGF-β1 in gingival fibroblasts. In addition, the signal transduction of CsA-induced gingival fibroblast proliferation was investigated.
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MATERIALS & METHODS
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All methods, including the collection and processing of the gingival samples, were approved by the IRB of the University of Wonkwang, South Korea. Written consent was obtained from each participant.
Materials
The cyclosporine A (CsA) was obtained from Sigma (St. Louis, MO, USA). The anti-p38 and GST-MBP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The PD98059, SB203580, and LY294002 were obtained from Calbiochem (La Jolla, CA, USA). The IL-6 and TGF-β1 immunoassay kits were obtained from Biosource International, Inc. (Camarillo, CA, USA). All the cell culture media and reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD, USA).
Cell Culture
Human gingival fibroblasts were prepared as described elsewhere (Wang et al., 2003). Briefly, the fibroblasts were harvested from four medically healthy donors, who were free of periodontal disease. The healthy tissues appeared firm, non-erythematous, non-edematous, and non-bleeding. The gingival tissue was surgically excised from adjacent to the maxillary tuberosity and washed 6 times with Hank’s solution. The minced gingival tissues were seeded in six-well plates with Dulbecco’s Modified Eagle’s medium supplemented with 2 mM L-glutamine and 10 w/v% heat-inactivated fetal bovine serum. The cells between passages 5 and 12 were used.
Assay for IL-6 and TGF-β1
ELISA assays were performed for IL-6 and TGF-β1 by a modification of the procedure reported elsewhere (Chae et al., 2001; Agarwal et al., 2002). After the human gingival fibroblasts were treated with various agents, the conditioned medium was collected, and IL-6 or TGF-β1 in the medium from the human gingival fibroblasts was then measured with the use of a human IL-6 or TGF-β1 ELISA kit. However, no serum-containing media were used in the experiments for neutralizing the endogenous IL-6 or TGF-β1, because a small amount of serum could capture the cytokines.
Immunoprecipitation and Kinase Assays
The p38 MAP and PI3Kinase assay and immunoblotting were performed by a modification of the procedure reported elsewhere (Linassier et al., 1997; Chae et al., 2001). For the p38 MAPK assay, the cells underwent lysis, and the supernatants were incubated with anti-p38 for 2 hrs at 4°C. The immunocomplexes were precipitated, and the precipitated pellets were dispersed as a 1:1 suspension in an assay buffer. Subsequently, a 20-µL (0.3 mg/mL) quantity of MBP was added. We determined the kinase activity by measuring the level of MBP phosphorylation.
Phosphatidylinositol 3-kinase Assay
Phosphatidylinositol 3-kinase assays were performed directly on the anti-phosphotyrosine immunoprecipitates in a 50-µL reaction mixture containing 0.2 mg/mL phosphoinositol, 50 µM ATP, 0.2 µCi [ -32P] ATP, 5 mM MgCl2, and 10 mM HEPES buffer (pH 7.5). The reactions were carried out for 15 min at room temperature and quenched by the addition of 100 µL of 1 N HCl and 200 µL of chloroform/methanol (1/1, V/V). The lipids were extracted and resolved on oxalate-coated thin-layer chromatography (TLC) plates (silica gel 60), with a 1-propanol/2 M acetic acid (65/35, V/V) solvent system. The oxalate-coated TLC plates were dried and subjected to autoradiography.
Northern Blotting
For Northern blotting, the total cellular RNA was prepared with TRIzol reagent (Gibco BRL, Gaithersburg, MD, USA), according to the manufacturer’s instructions. The RNA (20 µg) was subjected to electrophoresis in 1 w/v% formaldehyde agarose gels, and transferred to a nylon membrane. Hybridization was carried out at 42°C in 50 mM Tris-HCl, pH 7.4, 40 v/v% formamide, 4 SSC (15 mM sodium citrate, 150 mM NaCl), 10 Denhardt’s solution, 0.1 w/v% Na4P2O7, 1 w/v% SDS, and 200 µg/mL herring sperm DNA. The blots were washed and exposed to x-ray film at –70°C. Fragments of the hIL-6 and hTGF-β cDNAs were labeled with  -32P with random primers (Boehringer, Mannheim. Germany), and used as hybridization probes.
Immunoblotting
Western blotting was performed as described in the literature (Chae et al., 2001). The protein samples (cell extracts, 50 µg) were separated by SDS-PAGE and blotted onto a PVDF membrane. The membrane was blocked with 5 w/v% bovine serum albumin, 1 w/v% milk powder in 10 mM Tris-HCl containing 150 mM NaCl, and 0.5 w/v% Tween-20 for 1 hr, and incubated overnight with suitably diluted primary antibodies. After being washed, the filters were incubated with a peroxidase-conjugated secondary antibody for 1 hr before the immunolabeled bands were detected by ECL (Amersham Life Science, Piscataway, NJ, USA).
Statistical Analysis
The data from the dose-response experiments were evaluated by analysis of variance (ANOVA) and two-tailed Student’s t tests.
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RESULTS
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CsA, IL-6, or TGF-β1 Increases the Proliferation of Human Gingival Fibroblasts
Direct cell-counting of the human gingival fibroblasts incubated with or without CsA, IL-6, or TGF-β1 for 3, 6, or 9 days revealed that the level of proliferation was much higher in the presence of these agents. One hundred and 500 ng/mL CsA increased the level of gingival fibroblast proliferation, with the maximal stimulation being observed at 500 ng/mL (Fig. 1A ). Therefore, all subsequent studies were performed at a CsA concentration of 500 ng/mL. IL-6 and TGF-β1 also produced a significant increase in the rate of human gingival fibroblast proliferation relative to the control. A significant increase in cell proliferation was observed at IL-6 concentrations of 0.1 and 0.2 ng/mL. The TGF-β1 concentration where cell proliferation was observed ranged from 0.1 to 0.5 ng/mL (Fig. 1A). The effects of IL-6 and TGF-β1 on human gingival fibroblasts were also determined by bromodeoxyuridine-labeling (Fig. 1B). The mitotic-index derived from bromodeoxyuridine-labeling (Brdu) of the cells treated with CsA, IL-6, or TGF-β1 was also significantly higher than those of the controls. Serum starvation for 3 days resulted in the accumulation of more than 90% of the cells in the G0/1 phase of the cell cycle. The addition of CsA (100 or 500 ng/mL), IL-6 (0.1 or 0.2 ng/mL), or TGF-β1 (0.1 or 0.5 ng/mL) to the serum-starved cells caused re-entry into the cell cycle as well as the stimulation of DNA synthesis (Appendix Fig. 1). We used a neutralizing antibody to inhibit cell proliferation, to determine if CsA regulates the proliferation via TGF-β1 or IL-6 autocrine stimulation. It appears that both TGF-β1 and IL-6 are key factors in CsA-induced gingival proliferation (Fig. 1C). These results showed that CsA increases the levels of biologically active IL-6 and TGF-β1 in the supernatant in a time-dependent manner (Fig. 1D). In addition, the level of IL-6 mRNA and protein synthesis correlated with the release of the IL-6 cytokine within 24 hrs, as determined by Northern blotting and immunoblotting (IL-6) and ELISA (TGF-β1) (Appendix Figs. 2, 3).
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Figure 1. . IL-6 and TGF-β1 play an important role in CsA-induced proliferation in human gingival fibroblasts. (A) The cells were exposed to CsA (100 or 500 ng/mL), IL-6 (0.1 or 0.2 ng/mL), or TGF-β1 (0.1 or 0.5 ng/mL) for 0, 3, 6, or 9 days, and were counted (mean ± SD; n = 5). (B) The cells were stimulated with 100 or 500 ng/mL CsA, 0.1 or 0.2 ng/mL IL-6, or 0.1 or 0.5 ng/mL TGF-β1 for 6 days. The cells were then labeled with bromodeoxyuridine at four-hour intervals. The number of cells incorporating bromodeoxyuridine was counted (mean ± SD; n = 4). (C) The cells were treated with 500 ng/mL CsA, CsA+IL-6 neutralizing antibody (0.1 µg/mL), or CsA+TGF-β1 neutralizing antibody (1 µg/mL) (mean ± SD; n = 4). (D) After the 500 ng/mL CsA treatment, the IL-6 and TGF-β1 levels in the supernatants were measured (mean ± SD; n = 4). (B–D) *Significantly different from the control, p < 0.05. #Significantly different from CsA-treated, p < 0.05.
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Figure 2. CsA increased the activation of ERK, p38 MAPK, and PI3K in cells. (A) The cells were treated with 500 ng/mL CsA. Phosphorylated ERK1/2 and JNK1 levels were examined (a). The cells were treated with 500 ng/mL CsA in the presence or absence of 10 µM SB203580 (b). (B) CsA (500 ng/mL) was added for 5, 10, 20, 30, or 60 min. A PI3K activity assay was performed. (C) The cells either were untreated or were treated for 30 min with the indicated agents, followed by 24 hrs of stimulation with 500 ng/mL CsA. The IL-6 and TGF-β1 levels in the supernatant were then measured (mean ± SD; n = 4). (D) The cells were treated with the indicated agents for 30 min, followed by stimulation with 500 ng/mL CsA for 3 days, and were then counted (mean ± SD; n = 6). *Significantly different from the CsA-treated, p < 0.05.
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Figure 3. . TGF-β1 is a key factor in CsA-induced IL-6 release in human gingival fibroblasts. (A) The cells either were untreated or were treated for 30 min with TGF-β1 (1 or 2 µg/mL) or IL-6 neutralizing antibody (0.1 µg/mL), followed by 24 or 48 hrs of stimulation with 500 ng/mL CsA. The supernatants were assayed for IL-6 (mean ± SD; n = 5). (B) The cells were treated with the IL-6 (0.1 or 0.2 µg/mL) or TGF-β1 (1 µg/mL) neutralizing antibody, followed by 24 hrs of stimulation with 500 ng/mL CsA. The amount of TGF-β1 in the supernatants was measured (mean ± SD; n = 3). *Significantly different from the CsA-treated, p < 0.05. (C) For the IL-6 assay, the cells were treated with 1, 5, or 10 ng/mL TGF-β1 for 24 hrs (mean ± SD; n = 3). *Significantly different from the control, p < 0.05. #Significantly different from the CsA-treated, p < 0.05. (D) For the TGF-β1 assay, the cells were incubated with 0.1, 0.5, or 1 µg/mL IL-6 for 24 hrs (mean ± SD; n = 3).
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CsA Induces ERK, p38 MAPK, and PI3K Activations in Human Gingival Fibroblasts
The activation of MAPKs (ERK, p38, and JNK/SAPK) and PI3Kinase has previously been shown to be indispensable for the induction, differentiation, and proliferation of the IL-6 gene (Chae et al., 2001; Cotrim et al., 2003; Hallmann et al., 2003; Valverde et al., 2003; Fan et al., 2004). The CsA treatment activated ERK 1/2 or p38 MAP kinase but not JNK (Fig. 2A ). Incubation with CsA (500 ng/mL) for 5, 10, 20, or 30 min increased the production of phosphatidylinositol-phosphate (PI-P). PI-P is a substrate for PI3K activation and AKT, which is known to be a PI3K down-stream kinase (Fig. 2B, Appendix Fig. 4).
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Figure 4. Schematic diagram showing the signaling mechanism in CsA-induced gingival fibroblast proliferation.
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To evaluate the biological significance of ERK, p38, JNK, or PI3K activation by CsA, we examined the effects of SB203580 (a p38 MAPK inhibitor), PD98059 (a MEK1 inhibitor), SP600125 (a JNK/SAPK inhibitor), and LY294002 (a PI3K inhibitor) on the CsA-induced release of IL-6 and TGF-β1. Both the SB and PD compounds regulated the CsA-associated release of IL-6, and SB and LY 294002 abrogated the stimulation of TGF-β1 (Fig. 2C). The regulation of IL-6 and TGF-β1 mRNA and protein synthesis correlated with the data shown in Fig. 2C (Appendix Figs. 5, 6). We used specific inhibitors to block the MAPK or PI3K kinases in the CsA-treated cells, to determine if CsA regulates cell proliferation via MAPK or PI3K. Treatment with either SB203580 or LY294002 abrogated the CsA-induced proliferation of human gingival fibroblasts (Fig. 2D). It was demonstrated that both p38 MAPK and PI3K are essential in CsA-induced proliferation in the gingival fibroblasts. In addition, ERK might be involved in CsA-associated gingival proliferation. This suggests that both p38 and ERK MAPK are involved in the CsA-induced IL-6 release, and that p38 and PI3K are associated with TGF-β1 release, which can explain the CsA-induced gingival proliferation.
CsA Releases TGF-β1, Which Stimulates the Release of IL-6 in Human Gingival Fibroblasts
Although CsA increases both TGF-β1 and IL-6 in gingival tissues, it seems unlikely that the critical role of the cytokines and the related signaling pathways for this side-effect have not been well-studied (Myrillas et al., 1999; Buduneli et al., 2001; Cotrim et al., 2003). In this study, the 2 cytokines were found to play an important role in CsA-associated proliferation. Therefore, this study examined the order of the 2 cytokines, upstream or downstream, in human gingival fibroblasts. We used neutralizing antibodies specific to TGF-β1 and IL-6 to block the production of the cytokines by the CsA-treated human gingival fibroblasts, to show that CsA regulates cell proliferation via TGF-β1 or IL-6 autocrine stimulation. The addition of the IL-6 neutralizing antibody had a regulatory effect on IL-6 in the presence of CsA, but not on the release of TGF-β1 (Figs. 3A, 3B). In contrast, the cells treated with the TGF-β1 neutralizing antibody showed significantly decreased amounts of IL-6, in response to stimulation by CsA (Fig. 3A). These results show that CsA can induce the release of TGF-β1 and, subsequently, the release of IL-6 in human gingival fibroblasts. TGF-β1 directly stimulated IL-6 in human gingival fibroblasts, whereas IL-6 had no effect on the release of TGF-β1 (Figs. 3C, 3D). Furthermore, treating the fibroblasts with actinomycin D, which inhibits transcription, inhibited the production of IL-6 by TGF-β1 (Fig. 3C). Therefore, it is quite likely that the production of IL-6 by TGF-β1 is the result of de novo synthesis.
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DISCUSSION
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IL-6 is a multifunctional regulator of the immune responses, hematopoiesis, and various acute-phase reactions. In addition, it has also been shown to regulate cell hyperplasia (Badache and Hynes, 2001). TGF-β1 is a well-known growth factor that plays an important role in cell proliferation-associated pathophysiological states, such as gingival overgrowth (Cotrim et al., 2003). This study demonstrated that CsA up-regulates IL-6 and TGF-β1 gene expression in human gingival fibroblasts, suggesting a relationship between these 2 proteins and cell proliferation. These results suggest that the release of IL-6 in human gingival fibroblasts is dependent on TGF-β1. This was confirmed by pre-treatment of the cells with a TGF-β1 neutralizing antibody, which significantly reduced the release of IL-6 in the CsA-exposed cells (Fig. 3A). However, the IL-6 neutralizing antibody had no effect on the CsA-induced release of TGF-β1, suggesting that TGF-β1 contributes to the CsA-associated release of IL-6 in gingival proliferation. This is consistent with reports showing that TGF-β1 activates IL-6, which has been implicated in tumor behavior. The study also examined the important role of p38 MAPK signaling in the TGF-β1-induced release of IL-6 (Park et al., 2003).
In this study, p38 MAPK and PI3K were shown to play an important role in the release of IL-6 and TGF-β1, in addition to proliferation in CsA-exposed human gingival fibroblasts. We examined the effect of each kinase on CsA-induced gingival proliferation, to determine the biological significance of p38 MAP kinase, ERK MAP kinase, or PI3K activation by CsA. CsA-induced proliferation was inhibited particularly by the inhibition of p38 or PI3K (Fig. 2D). In addition, this study showed that the ERK1/2 MAPK signaling pathway is responsible for the release of IL-6, but is only partially responsible for cell proliferation (Figs. 2A, 2C, 2D). All of the signaling pathways examined in this study revealed that CsA could increase the release of both cytokines, IL-6 and TGF-β1, in human gingival fibroblasts. These results are in contrast to those in a recent report, which demonstrated that CsA treatment significantly reduced serum IL-6 levels (Myrillas et al., 1999). However, their study used human peripheral blood mononuclear cells, while this study used human gingival fibroblasts.
It is well-known that PI3K signaling is involved in many cellular processes, including proliferation (Zhao et al., 2001). In contrast, p38 MAPK, which is a well-known inflammatory MAPK, has not been examined in the field of proliferation. However, McGinn et al. (2003) suggested that TGF-β1 increases the levels of phosphorylated p38 MAP kinase, and that the p38 MAP kinase blockade reversed the anti-proliferative effects of this cytokine. In addition, their study also showed that p38 MAPK can have an important effect on proliferation. In concurrence with their study, the present study suggests that both p38 MAP kinase and PI3K are essential for CsA-induced proliferation. In human gingival fibroblasts, p38 MAPK is a key pathway for immunosuppressant-associated IL-6 production, and both p38 MAPK and PI3K play an important role in TGF-β1 production (Fig. 4). This signal transduction pathway suggests a strategy for regulating the CsA-induced TGF-β1 and IL-6 production, and the subsequent gingival proliferation and overgrowth.
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ACKNOWLEDGMENTS
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This work was supported by a research grant from the Korea Research Foundation (Grant KRF-2000-005-F00001).
Received for publication June 8, 2005.
Revision received March 1, 2006.
Accepted for publication March 20, 2006.
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Journal of Dental Research, Vol. 85, No. 6,
515-519 (2006)
DOI: 10.1177/154405910608500607
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ABSTRACT
INTRODUCTION