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Effect of interleukin-33 on cementoblast-mediated cementum repair during orthodontic tooth movement
Journal Pre-proof Effect of interleukin-33 on cementoblast-mediated cementum repair during orthodontic tooth movement Xiaomeng Dong, Jie Feng, Ji Wen, Ding Bai, Hui Xu
PII:
S0003-9969(19)31184-7
DOI:
https://doi.org/10.1016/j.archoralbio.2020.104663
Reference:
AOB 104663
To appear in:
Archives of Oral Biology
Received Date:
22 November 2019
Revised Date:
4 January 2020
Accepted Date:
9 January 2020
Please cite this article as: Dong X, Feng J, Wen J, Bai D, Xu H, Effect of interleukin-33 on cementoblast-mediated cementum repair during orthodontic tooth movement, Archives of Oral Biology (2020), doi: https://doi.org/10.1016/j.archoralbio.2020.104663
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Effect of interleukin-33 on cementoblast-mediated cementum repair during orthodontic tooth movement
Xiaomeng Dong a, Jie Feng a, Ji Wen a, Ding Baib, Hui Xub,* a State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, Sichuan University, Chengdu, China. b State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases;
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Department of Orthodontics, West China Hospital of Stomatology; Sichuan University, Chengdu, China.
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*Corresponding author: Hui Xu telephone: 86-028-85501474
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e-mail: [email protected]
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address:Department of Orthodontics, West China Hospital of Stomatology, 3rd section of South Renmin Road,
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Chengdu, China, 610041.
Highlights
Heavy orthodontic force increases interleukin-33 expression in periodontal
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tissue.
ST2 is expressed in cementoblast-like cells and is regulated by interleukin-33.
Interleukin-33 abates differentiation and mineralization of OCCM-30 cells.
Interleukin-33
abates
cementogenesis-related
protein
expressions
in
OCCM-30 cells.
Interleukin-33 down-regulates the ratio of osteoprotegerin/RANKL in 1
OCCM-30 cells.
Abstract Objective: This study aims to uncover the role of interleukin-33 on cementoblast-mediated cementum repair. Methods: 6-8-week-old C57BL/6 mice were used to establish the model of orthodontic tooth movement.
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Interleukin-33 and suppression of tumorigenicity2 (ST2) expressions were
immunohistochemically detected in the periodontal tissue. In vitro, cementoblast-like (OCCM-30) cells were cultured in the presence of recombinant mouse interleukin-33 protein (rmIL-33) at a
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1-14 d time frame. ST2 expressions were immunofluorescently labeled and quantitatively
examined. The effects of interleukin-33 on cementoblast differentiation, mineralization and
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proliferation were examined by alkaline phosphatase, alizarin red staining and cell counting kit-8, respectively. To further clarify the effect of interleukin-33 on cementogenesis-related protein
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expressions, runt-related transcription factor 2 (RUNX2), osterix, osteopontin, bone sialoprotein(BSP), osteocalcin, osteoprotegerin (OPG) and receptor activator of NF-КB ligand
Results:
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(RANKL) expressions were examined by western blot.
Orthodontic load of high magnitude induces external apical root resorption, and increases
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interleukin-33 expression in the periodontal tissue of mice. Cells in the cementum express ST2. Interleukin-33 initially down-regulates but later recovers ST2 mRNA and protein levels in
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OCCM-30 cells. Interleukin-33 abates cementoblast differentiation and mineralization, and suppresses RUNX2, osterix, BSP and osteopontin expressions in OCCM-30 cells at the later stage of the culture period. Interleukin-33 enhances RANKL expression, and reduces the ratio of OPG/RANKL in OCCM-30 cells. Conclusion: Orthodontic load of high magnitude induces interleukin-33 expression in the periodontal tissue. Interleukin-33 has a negative effect on cementogenesis via suppressing cementoblast
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differentiation, mineralization and cementogenesis-related protein expressions.
Keywords: interleukin-33, cementoblast, cementum, tooth movement
Introduction External apical root resorption, characterized as a pathological process that brings about resorption of the superficial root cementum, is a frequent side effect secondary to orthodontic treatment (Feller, Khammissa, Thomadakis, Fourie, & Lemmer, 2016; Fernandes et al., 2019;
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Iglesias-Linares & Hartsfield, 2017; Nieto-Nieto, Solano, & Yanez-Vico, 2017). Teeth may also suffer external apical root resorption in the absence of orthodontic treatment, in response to
traumatic, inflammatory, autoimmune or infectious stimuli (Nieto-Nieto et al., 2017). External apical root resorption had been reported to occur in over 90% of orthodontic patients, among
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which, aggressive external apical root resorption (defined as a loss of length exceeding 5 mm) has been seen in 2–5% of orthodontic patients (Feller et al., 2016; Fernandes et al., 2019;
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Iglesias-Linares & Hartsfield, 2017; Nieto-Nieto et al., 2017). However, loss of root length is clinically insignificant in the majority of cases, owing to root remodeling and repair occurring at
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the cellular cementum (Feller et al., 2016).
Dental root remodeling is a dynamic process during which cementum resorption and subsequent
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repair by cellular cementum take place (Feller et al., 2016; Iglesias-Linares & Hartsfield, 2017).Biologically, the susceptibility of the dental root to pathologic resorption is dictated by the
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counterbalance between two key cellular aspects, one is formation and activation of cementoclasts, the other is cementoblast-mediated remineralization and cementogenesis which account for repair
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capabilities (Feller et al., 2016; Iglesias-Linares & Hartsfield, 2017). Signals orchestrating these cellular aspects have the potential to influence the extent and frequency of external apical root resorption, but the molecular and cellular mechanisms which mediate this process are poorly understood. External apical root resorption results from multifactorial causes including intrinsic factors, environmental factors and variables related to treatment (Al-Qawasmi et al., 2003; Nieto-Nieto et al., 2017). Previous studies emphasized intrinsic causes as important etiological factors by 3
reporting that genetic predisposition accounted for at least 50% of variation in post-orthodontic external apical root resorption, with variations in the interleukin-1B gene determining 15% of the differences (Hartsfield, Everett, & Al-Qawasmi, 2004), though it is still unclear how interleukin-1 gene determines the predisposition of root resorption. Interleukin-33 (IL-33) was identified as a member of theinterleukin-1 family and a ligand for the receptor suppression of tumorigenicity2 (ST2) (Liew, Pitman, & McInnes, 2010; Schmitz et al., 2005). IL-33 is constitutively expressed in structural and lining cells, and is released into the extracellular space upon tissue damage and/or mechanical injury (Xu, Turnquist, Hoffman, &
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Billiar, 2017). IL-33 is recognized to act as an alarmin to sense damage and alert neighboring cells following infection or trauma, and has potent influence on a broad range of diseases by targeting various cell types (Kurowska-Stolarska, Hueber, Stolarski, & McInnes, 2011; Palmer & Gabay,
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2011; H. Xu et al., 2017; Xu et al., 2018; J. Xu et al., 2017). The role of IL-33 on cementogenesis and dental root remodeling is unexplored. This study aims to investigate the role of IL-33 in
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cementoblast-mediated cementum repair during orthodontic tooth movement.
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Materials and Methods Animals
All protocols in this study were approved by the Bioethics Committee of Sichuan University. 10
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male C57BL/6 mice (Dashuo, Sichuan, China) were obtained and bred in a temperature-controlled room with a 12/12-hour light–dark cycle. All mice were used at the age of 6–8 weeks.
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Orthodontic tooth movement model
We used a split-mouth method for the orthodontic tooth movement model. After the mice were
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anesthetized by an intraperitoneal injection of 10% chloral hydrate (0.3mg/kg), an closed-coil spring (Mingxin, Chengdu, China) was activated and fixed between the maxillary first molar and maxillary incisor on the right side, to exert a force of 40 g initially without re-activation or interruption during 14 days. The magnitude of force was chosen at a high level to mimic the condition when heavy orthodontic load was applied. (Wolf et al., 2018). The contralateral teeth on the left side were also ligated to the appliance but without activation of the coil spring, serving as vehicle control. The teeth of mice were subjected to orthodontic load or to unloaded control for 14 4
days. Mice were euthanized by an overdose of CO2 inhalation at 14 d. The maxillas were removed and fixed in 4% paraformaldehyde for 48h at 4℃, then were decalcified in 10% disodium ethylenediaminetetraaceticacid (EDTA, pH 7.4) solution for 1 month. After decalcification, the maxillary tissues were dehydrated and embedded in paraffin. 4-μm-thicksections were prepared for experiments.
Immunohistochemistry and Hematoxylin and eosin (HE) staining.
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The tissue sections were dewaxed, rehydrated, then incubated with 0.1% trypsin for 30min at 37℃ for antigen retrieval. The sections were stained with Hematoxylin and eosin for observation of histological changes. Immunohistochemical staining was performed using a mouse/rabbit
streptomyces oval-biotin assay (Zhongshan Golden Bridge Biotechnology, Beijing, China). Briefly,
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the sections were blocked with 3% hydrogen peroxide for 40 min, blocked with 1% BSA for
30min and then incubated with anti-IL33 (1:600, Abcam, USA) or anti-ST2 primary antibodies
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(1:600, Abcam, USA) overnight at 4℃. After being incubated with biotin-labeled goat anti-mouse/rabbit IgG polymer for 10 min and then with horseradish-labeled streptomycin for
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10min at room temperature, the reaction products were visualized with a diaminobenzidine substrate kit (Zhongshan Golden Bridge Biotechnology, Beijing, China). The sections were
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counterstained with haematoxylin or methyl green (Solarbio, Beijing, China). Cell culture and drug administration
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Cementoblast-like (OCCM-30) cells, a murine cell line, were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China) and cultured in alpha-Minimum Essential
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Medium (α-MEM) (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% streptomycin/penicillin (Gibco, USA) as described previously(Oka et al., 2007).After reaching 80% confluence, cell growth was arrested by incubating in serum-free medium for 24 hours. Afterwards, the quiescent cells were cultured in α-MEM with 5% FBS. For drug administration, recombinant mouse interleukin-33 (rmIL-33, R&D, USA) was added to the culture medium (α-MEM plus 5% FBS), at a concentration of 5ng/ml or 20ng/ml. For vehicle control, the culture medium was supplied with phosphate-buffered saline (PBS), instead of recombinant protein. 5
The medium was replaced with fresh medium every other day, to maintain a relatively stable drug concentration during a long culture period. The cultures were divided into five groups. OCCM-30 cells in each group were subjected to rmIL-33 stimulation or vehicle control for 1,3,5,7,14days, respectively. Cells were harvested at indicated time point during the 1-14 d time frame. Experimental procedures were repeated no less than three times for each experimental condition.
Immunofluorescence OCCM-30 cells were seeded onto 4-well glass slides at a density of 4x10^3 cells/well (Millipore,
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USA) for Immunofluorescent labeling of ST2 expression. After being fixed with 4% paraformaldehyde for 20min, the cells were permeabilized with 0.1% triton for 8min and then
blocked with 1% BSA (Biofroxx, Germany) for 30min. The cells were incubated with anti-ST2 primary antibody (1:200, Abcam, USA) for 16h at 4℃, and then incubated with goat anti-rabbit
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IgG antibody (1:50, Bioss, China) for 1 h at 37℃.The nuclei were stained with
Cell Counting Kit-8 (CCK-8)
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fluorescence microscope (Leica, USA).
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4,6-diamidino-2-phenylindole(DAPI) (solarbio, Beijing, China).Images were captured by a
OCCM-30 cells were seeded into 96-wellplates at a density of 4x10^3 cells/well. After being
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cultured overnight, the cells were treated with rmIL-33 (5ng/ml, 20ng/ml) or vehicle control for 1,3,5,7 days, respectively. The cells were washed with PBS, then incubated with culture medium supplemented with 10% CCK-8 (Dojindo, Japan) for 30 min at 37℃. The optical density (OD)
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values were read at 450nm wavelength.
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Alkaline phosphatase (ALP) staining and quantitative analysis OCCM-30 cells were seeded into 12-well plates at a density of 5x10^4cells/well. After being cultured overnight, the cells were treated with rmIL-33 (5ng/ml, 20ng/ml)or vehicle control for 7 days. Cells were stained using BCIP/NBT alkaline phosphatase color development kit (Beyotime, Beijing, China). ALP activity was quantified by alkaline phosphatase assay kit (Beyotime, Beijing , China).
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Alizarin red staining (ARS) and quantitative analysis OCCM-30 cells were seeded into 12-well plates at a density of 5x10^4 cells/well. After being cultured overnight, the cells were cultured in osteogenic differentiation medium, that is supplemented with10^8mmol/L dexamethasone (Sigma, USA), 0.2mmol/L ascorbic acid (J&K, Beijing, China) and10mmol/L β-glycerophosphate (Sigma, USA), with or without rmIL-33 (0ng/ml, 5ng/ml, 20ng/ml) for 21days. After being washed with PBS, the cells were incubated with 0.1% alizarin red solution for 10 min (PH=4.3, Solarbio, Beijing, China). After being captured by a camera, the mineralized nodules were dissolved in 2ml 10% cetylpyridinium
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chloride (J&K, Beijing, China), then the OD values were read at 562nm wavelength.
Quantitative Real-time PCR (qRT-PCR)
OCCM-30 cells were seeded into 6-well plates at a density of 20x10^4 cells/well, and treated with
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rmIL-33 (5ng/ml, 20ng/ml) or vehicle control for 1,3,5,7,14 days, respectively. The total RNAs were extracted with trizol reagent (Thermo, USA). The 1μgcomplementary DNA (CDNA)
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synthesis was perfomed using PrimeScript™ RT reagent Kit (TaKaRa, Japan). The premier sequence:ST2 (17082):5’-GCCACAGGACATCAGCCAAGAAG-3’ (forward) and 5’-GA-
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CGGCCACCAGATCATTCACAG-3’ (reverse); GAPDH (14433):5’-AGGTCGGTGTGAACGGATTTG-3’ (forward) and 5’-TGTAGACCATGTAGTTGAGGTCA-3’ (reverse).QRT-PCR was
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performed using TB Green® Fast qPCR Mix (TaKaRa, Japan) in Quant Studio™3 Flex Real-time PCR System. The ST2 relative mRNA expression was calculated using the 2 ^-ΔΔCt method.
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Western blot assay
OCCM-30 cells were seeded into 6-well plates at a density of 20x10^4 cells/well, and treated with
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rmIL-33 (5ng/ml, 20ng/ml) or vehicle control for 1, 3, 5, 7, 14 days, respectively. Proteins were extracted with a protein extraction kit (SAB, USA). The protein concentration were measured using a BCA protein assay kit (Beyotime, Bijing, China). Equal amounts of protein extracts were separated on 10% SDS–polyacrylamide gels (Biorad, USA), and then transferred onto polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked in 5% skim milk (Biofroxx, Germany) for 1h at room temperature, then incubated with rabbit anti-runt-related transcription factor 2 (RUNX2) (1:1000,Huaan, Shanghai, China), rabbit anti-osteopontin 7
(1:1000,Proteintech, USA), rabbit anti-osterix (1:1000, Abcam, USA), mouse anti-bone sialoprotein (BSP) (1:300, Santa Cruz, USA), rabbit anti-osteocalcin
(1:1000, Affinity, USA),
rabbit anti-osteoprotegerin (OPG) (1:1000, Affinity, USA), rabbit anti-receptor activator of NF-КB ligand (RANKL) (1:1000, Affinity, USA), for 16h at 4℃. After being washed with TBST, the membranes were incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG (SAB, USA) and visualized with chemiluminescent substrate (US Everbright lnc, USA). Quantitation of the protein levels were analyzed by Image J software (National Institutes of Health,
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Bethesda, MD).
Statistical analysis
All data were analyzed with Graphpad Prism 7.0 (Graphpad Software Inc, San Diego, CA).
Experimental results were presented as mean ± SD of triplicate determinations. Unpaired t-test
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was used for comparison of two groups. Analysis of variance (ANOVA) were used for comparison among groups, and Tukey's multiple comparison test were used for multiple comparisons between
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pairs. A probability at the 5% level or less (P < 0.05) was considered statistically significant.
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Results
Orthodontic force of high magnitude leads to increased IL-33 expressions in the periodontal
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tissue and induces root resorption in mice
We examined interleukin-33 and ST2 expressions in the periodontal tissue under orthodontic load
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(figure 1). Immunohistochemistry staining revealed increased IL-33 expression in compression areas of the PDL under 40 g orthodontic force, compared with unloaded control (figure 1A).
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Immunostaining for ST2 was positive in the periodontal tissue and in the dental pulp of mice, irrespective of orthodontic loading or unloaded control (figure 1B). Interestingly, ST2 expression was observed in cells in the cementum (figure 1B). It is well recognized that orthodontic forces of high magnitude cause root resorption, and we confirmed this by finding resorption lacunae in compression areas of the root surface under 40 g orthodontic force in a mouse model (figure 1B, C). The concomitant occurrence and adjacent location of root resorption and IL-33/ST2 expression lead us to investigate the role of IL-33 in cementum remodeling. 8
Cementoblast-like (OCCM-30) cells express ST2. IL-33 initially down-regulates but later recovers ST2 mRNA and protein levels in OCCM-30 cells. Immunofluorescence showed ST2 expression in OCCM-30cells (figure 2A). We also examined gene and protein levels of ST2 expression by qRT-PCR (figure 2B) and western blot (figure 2C). To test whether ST2 expression is regulated by its ligant, OCCM-30 cells were cultured in the presence of different concentrations of rmIL-33 protein. The expression levels of ST2 were quantitatively examined at 1-14d time frame during the culture period. ST2 mRNA levels in OCCM-30 cells were down-regulated at 3-7d (figure 2B, P<0.05), and recovered at 14 d in the
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presence of 5ng/ml or 20ng/ml rmIL-33. Similar trend was found for the change of ST2 protein
levels over time in the presence of rmIL-33 stimulation, with a significant up-regulation of ST2 by 20ng/ml rmIL-33at 14 d (figure 2C, P<0.05).
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IL-33 abates cementoblast differentiation and mineralization of OCCM-30 cells
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We investigated the effects of IL-33 on OCCM-30 cell differentiation by ALP staining and quantitative analysis (figure 3A). ARS and quantitative analysis were performed to detect the
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effect of interleukin-33on cell mineralization (figure 3B). rmIL-33 inhibited cementoblast differentiation of OCCM-30 cells at a concentration of 5ng/ml or 20ng/ml (figure 3A, P<0.05). Formation of calcium nodules were observed in OCCM-30 cell cultures at 21d. Treatment of
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5ng/ml or 20ng/ml rmIL-33 attenuated OCCM-30 cell mineralization, compared with vehicle control (figure 3B, P<0.05).
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The proliferative response of cementoblast-like cells to IL-33 was examined by a CCK-8 assay kit. As showed in figure 3C, 5ng/ml rmIL-33 had no effect on OCCM-30 cells proliferation at 1-7d
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time frame during the culture period, while 20ng/ml rmIL-33 didn’t significantly inhibited cell proliferation until 7 d (figure 3A, P<0.05). IL-33 suppresses expressions of RUNX2, osterix, BSP and osteopontin in OCCM-30 cells at the later stage of the culture period To further clarify the effect of IL-33 on cementogenesis-related protein expressions in OCCM-30
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cells, expressions of RUNX2, osterix, osteopontin, osteocalcin and BSP in OCCM-30 cells were examined by western blot (figure 4-5) at 1-14 d time frame under constant rmIL-33 stimulation. As presented in figure 4, administration of 5ng/ml rmIL-33 has no significant effect on expressions of RUNX2 and osteopontin at 1-7 d, but inhibited their expressions at 14 d (P<0.05). rmIL-33 inhibited osterix expressions as early as 3-5 d at a concentration of 20ng/ml (figure 4, P<0.05). rmIL-33 inhibited BSP expressions at 3 d, with a trend towards abating BSP levels at 14 d (figure 5A). 5ng/ml or 20ng/ml rmIL-33 didn’t significantly affect osteocalcin expressions in OCCM-30 cells at 1-14 d (figure 5B, P>0.05).
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IL-33 promotes RANKL expressions in OCCM-30 cells, and down-regulates the ratio of OPG/RANKL.
We investigated expressions of OPG and RANKL proteins in OCCM-30 cells over time under
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IL-33 stimulation, and calculated the ratio of OPG/RANKL. The time course analysis revealed a
trend towards initial suppression but later promotion of OPG expressions in OCCM-30 cells under
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rmIL-33 stimulation at a concentration of 5ng/ml or 20ng/ml (figure 6). In contrast, the change of RANKL expression showed a trend towards initial up-regulation, then down-regulation, followed
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by a significant increase at 14 d under rmIL-33 administration (figure 6). IL-33 down-regulated the ratio of OPG/RANKL in OCCM-30 cells at the 1-14 d time frame during the culture period,
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Discussion
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compared with vehicle controls (figure 6, P<0.05).
Orthodontic load-induced external apical root resorption was described as a complex, sterile
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inflammatory process that involves a dynamic interplay between local immune microenvironment and dental hard tissue(Feller et al., 2016). Inflammatory cytokines orchestrate this signaling network and influence the process of root resorption. Polymorphism of genes encoding cytokines and growth factors constitute an intrinsic factor in the pathogenesis of orthodontic load-induced external apical root resorption (Feller et al., 2016; Nieto-Nieto et al., 2017). Research groups have analyzed the genetic influence on the susceptibility to post-orthodontic external apical root resorption, such as interleukin-1 gene cluster, tumor necrosis factor-α, OPG, RANK and
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osteopontin gene, among which,interleukin-1 gene was highlighted by most studies(Al-Qawasmi et al., 2003; Bastos Lages et al., 2009; Gulden et al., 2009; Iglesias-Linares et al., 2012; Nieto-Nieto et al., 2017). The relationship between interleukin-1A or interleukin-1B gene polymorphisms and the risk of post-orthodontic external apical root resorption was determined (Bastos Lages et al., 2009; Gulden et al., 2009; Iglesias-Linares et al., 2012). Persons homozygous for allele 1 of the interleukin-1B gene have a 5.6-fold increased risk of post-orthodontic external apical root resorption compared with heterozygous subjects and those homozygous for allele 2 (Al-Qawasmi et al., 2003). Researchers tried to explain the mechanism by speculating that allele 1
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of interleukin-1B gene may cause reduced levels of interleukin-1β protein and impaired alveolar bone resorption, and thus result in prolonged stress concentrated around the adjacent
tooth root, without clarifying whether and how these genes have direct influence on the cementum
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(Hartsfield et al., 2004).
Root resorption is counterbalanced by cementum repair, otherwise would cause permanent loss of
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root length (Feller et al., 2016). Cementoblast-mediated mineralization and cementogenesis play vital roles in cellular cementum repair (Feller et al., 2016; Rego et al., 2011). Factors regulating
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cementogenesis determine the repair capabilities of the root, thus influencing the extent and frequency of external apical root resorption(Feller et al., 2016; Iglesias-Linares & Hartsfield, 2017). Previous studies demonstrated the association of root resorption with various cytokines
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including interleukin-1α, interleukin-1β,tumor necrosis factor-α and interleukin-6, though didn’t look into the mechanism whether and how these cytokines affect cementogenesis and root
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repair(Bastos et al., 2017; Kikuta, Yamaguchi, Shimizu, Yoshino, & Kasai, 2015; Kunii et al., 2013; Matsumoto, Sringkarnboriboon, & Ono, 2017; D. Zhang, Goetz, Braumann, Bourauel, &
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Jaeger, 2003). Limited evidence was from Diercke (Diercke, Konig, Kohl, Lux, & Erber, 2012), who presented that combination of compression and interleukin-1β stimulation impeded bone sialoprotein and cementum Protein 1 expression in human primary cementoblasts, while compression or interleukin-1β stimulation alone led to increased levels of bone sialoprotein and decreased levels of cementum Protein 1. In this study, we revealed IL-33, a member of the interleukin-1 family, as a negative regulator of cementogenesis via suppressing cementoblast differentiation and mineralization and cementogenesis-related protein expressions. IL-33 may 11
mediate impairment of cementum repair and consequently aggravate root resorption. IL-33 is an immunoregulatory cytokine which plays diverse and context-specific roles in various diseases (Kurowska-Stolarska et al., 2011; Palmer & Gabay, 2011; H. Xu et al., 2017). Recently, IL-33 was demonstrated to have interaction with skeletal system, though its role in osteogenesis remains elusive (Heckt et al., 2016; Kukolj et al., 2019; Mine, Makihira, Yamaguchi, Tanaka, & Nikawa, 2014; Saidi et al., 2011; Saleh et al., 2011). Cementum is similar to bone in many aspects, while cementum differs from bone in cellular components and having limited capacity for remodeling (Feller et al., 2016). It is controversial whether osteoblasts and cementoblasts are
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derived from a common precursor cell, though they both share many properties with respect to
phenotype, function and driven factors (Cao et al., 2015; Cao et al., 2012; Feller et al., 2016). It
was reported that IL-33 increases mRNA expression and secretion of RANKL in MC3T3-E1 cells
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(Mine et al., 2014), while induces expression of Tnfsf11 (RANKL-encoding gene)
in primary osteoblasts without soluble RANKL release (Heckt et al., 2016). Consistently, we
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showed increased expression of RANKL protein in OCCM-30 cells under IL-33 stimulation. Saleh(Saleh et al., 2011) demonstrated that IL-33 promoted matrix mineral deposition in primary
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osteoblasts in vitro. In contrast, we showed suppressed mineralization of OCCM-30 cells after IL-33 administration. These comparisons indicated that IL-33-driven responses are delicately
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cell-type-specific.
The IL-33 receptor ST2 is a member of the interleukin-1 receptor family (Kurowska-Stolarska et al., 2011; Palmer & Gabay, 2011; H. Xu et al., 2017). Membrane-bound ST2 is the functional
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component for IL-33 signaling (Kurowska-Stolarska et al., 2011; Palmer & Gabay, 2011; H. Xu et al., 2017). Observed ST2 expression in cells in the cementum and OCCM-30 cells indicated them
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as target cells of IL-33. Interestingly, we found that ST2 levels was initially down-regulated but later recovered by IL-33 in vitro, and in vivo, heavy orthodontic forces induced increased IL-33 expression with basal-level expression of ST2 in the periodontal tissue at corresponding time point, compared with unloaded control. It can be speculated that a negative feedback loop may exist between IL-33 and its receptor at early but not the later stage of culture period. Continuous IL-33 stimulation eliminated the negative feedback, and even drives a positive feedback at a high concentration, as we saw that 20ng/ml rmIL-33significantly up-regulated ST2 expression at 14 d 12
in vitro. Notably, the time frame when ST2 expression was recovered corresponds with that when cementoblast differentiation and cementogenesis-related protein expressions were suppressed. The regulation of ST2 expression by IL-33 may help explain the time-dependent change of effect of IL-33. Under IL-33 stimulation, the IL-33/ST2 signaling may be initially cancelled out by the negative feedback, and exert its power over time as the negative feedback disappeared. In the process of cementum repair, osteopontin and BSP are noncollagenous matrix proteins playing vital roles in formation of cementoid repair matrix at early stage of root repair (Feller et al., 2016; Jager et al., 2008). Osteopontin and osteocalcin contribute to the recruitment of
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cementoblast precursors to the root surface and to their subsequent adhesion, proliferation and differentiation(Feller et al., 2016; Jager et al., 2008). Mineralization ensues with cementoblast
proliferation and differentiation, forming reparative cementum and promoting cementogenesis
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(Feller et al., 2016). Working upstreams are the transcription factors RUNX2and osterix. RUNX2
is the master transcription factor that positively regulates the genes encoding alkaline phosphatase,
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collagen type 1,osteocalcin, and osterix (P. Zhang, Wu, Jiang, Jiang, & Fang, 2012). Osterix is a transcription factor functions molecularly downstream from RUNX2, and upregulates osteopontin,
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osteocalcin, and bone sialoproteingene and protein levels(Cao et al., 2015; Hakki et al., 2010). Osterixis essential for the differentiation of precementoblasts into mature cementoblasts (Cao et al., 2015; Feller et al., 2016). We investigated these cementogenesis-related protein expressions in
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cementoblast-like cells and found they were suppressed by interleukin-33 at not the early but the later stage of culture period, indicating that continuous IL-33 stimulation may cause impaired
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cementum repair.
Cautions should be taken against over-interpretation of the in-vitro effects on cementoblast-like
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cells, as biological properties of a cementoblast-like cell line is not identical to that of primary cementoblast culture. It is also worth noting that the in-vitro experiments cannot reproduce the multitude of factors in vivo. The in-vivo effects are derived from interactions among different types of cells and the extracellular matrix in the periodontal tissue. Further investigations are needed to explore the mechanism by which IL-33 suppresses differentiation and mineralization of cementoblasts, and to examine whether IL-33 has effects on other types of cells in the periodontal tissue. 13
In general, we revealed that orthodontic forces of high magnitude caused external apical root resorption in mice, and concomitantly induced increased IL-33 expression in the periodontal tissue. The IL-33 receptor ST2 was found expressed in the cementum and in cementoblast-like (OCCM-30) cells. IL-33 abated cementoblast differentiation and mineralization of OCCM-30 cells, inhibited expressions of RUNX2, osterix, osteopontin and BSP in these cells at the later stage of the culture period. IL-33 promoted RANKL expressions in OCCM-30 cells, and down-regulated the ratio of OPG/RANKL. These results indicated an anti-reparative effect of IL-33 on cementum remodeling under orthodontic load, mediated by suppressed cementoblast
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differentiation and mineralization and down-regulation of cementogenesis-related proteins.
Declaration of Competing Interest
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The authors declare no conflict of interest.
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Funding
This study was funded by National Nature Science Foundation of China (Grant Nos 81701006 and
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81870804).
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Conflict of interest
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The authors declare no conflict of interest.
Author Contributions: The conception and design of the study：Hui Xu, Xiaomeng Dong, Ding Bai Acquisition of data：Xiaomeng Dong, Jie Feng Analysis and interpretation of data: Xiaomeng Dong , Ji Wen Drafting the article: Xiaomeng Dong, Hui Xu 14
Revising it critically for important intellectual content: Hui Xu, Ding Bai, Jie Feng, Ji Wen, Final approval of the version to be submitted: Xiaomeng Dong, Jie Feng, Ji Wen, Ding Bai, Hui Xu
Acknowledgement The authors would like to acknowledge Bin Li for helping review and edit this manuscript, and
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acknowledge Chaoran Xue and Mengting He for data analysis.
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29(6), 1083-1089. doi:10.3892/ijmm.2012.934
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Figure Captions Figure 1. Heavy orthodontic force increases interleukin-33 expressions in the periodontal tissue and induces root resorption in mice. Representative images showing the expressions of interleukin-33 (A) and ST2 (B) in the periodontal tissue at 14 d under 40 g orthodontic force or unloaded control. The study areas were all selected around the distal roots of the maxillary first molars. →: the direction of tooth movement; scale bar: 100μm (upper panel), 50μm (lower panel). (C) Representative HE staining images of the periodontal tissue at 14 d under orthodontic load or
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unloaded control. The study areas were all selected around the distal roots of the maxillary first molars. →: the direction of tooth movement; scale bar: 200 μm (upper panel), 50 μm
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(lower panel).
Figure 2. Cementoblast-like (OCCM-30) cells express ST2. ST2expressions were initially down-regulated but later recoveredby interleukin-33 stimulation.(A) Representative 19
immunefluorescence staining for ST2 in cementoblast-like (OCCM-30) cells. Green: ST2, Blue: DAPI. Scale bar: 100μm. OCCM-30 cells were cultured in the presence of rmIL-33(5ng/ml, 20ng/ml) or as vehicle control for 1, 3, 5, 7, 14 days, respectively. Gene and protein levels of ST2 expressionswere quantitatively examinedby qRT-PCR (B) and western blot (C). Data were shown
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as Mean ± SD; (B) n=9 per group,(C) n=3 per group.*P<0.05, **P<0.01.
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Figure 3. Interleukin-33 abates cementoblast differentiation and mineralization of OCCM-30 cells. (A) Representative ALP staining of OCCM-30 cells treated with rmIL-33 (5ng/ml, 20ng/ml) or vehicle control for 7 days. The ALP activity was also quantitatively analyzed. Data were shown as Mean ± SD; n=9.*P<0.05, **P<0.01. (B)Representative ARS of OCCM-30 cells cultured in osteogenic differentiation medium with rmIL-33 (5ng/ml, 20ng/ml) or vehicle control for 21 days. The alizarin red activity was also quantitatively analyzed. Data were shown as Mean ± SD; n=9.*P<0.05, **P<0.01. (C)OCCM-30 cells were treated with rmIL-33 (5ng/ml,
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20ng/ml) or vehicle control for 1, 3, 5, 7 days, respectively. The proliferative activities of these
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cells were examined by CCK-8 assay. Data were shown as Mean ± SD; n=6.*P<0.05, **P<0.01.
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Figure 4. Interleukin-33 suppresses expressions of RUNX2, osterixand osteopontin in OCCM-30 cells at the later stage of the culture period. OCCM-30 cells were cultured with
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rmIL-33 (5ng/ml, 20ng/ml) treatment or as vehicle control for 1, 3, 5, 7, 14 days, respectively. Expressions of RUNX2 (A), osterix (B) and osteopontin (C) were quantitatively examined by western blot.Data were shown as Mean ± SD; n=3-8, *P<0.05, **P<0.01.
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Figure 5. Effect of interleukin-33 on the expressions of BSP and osteocalcin in OCCM-30 cells. OCCM-30 cells were cultured with rmIL-33 (5ng/ml, 20ng/ml) treatment or as vehicle control for 1, 3, 5, 7, 14 days, respectively. Expressions of BSP (A) and osteocalcin(B) were
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**P<0.01.
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quantitatively examined by western blot. Data were shown as Mean ± SD; n=3-6, *P<0.05,
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Figure 6. Interleukin-33 promotes RANKL expressions in OCCM-30 cells, and down-regulates the ratio of OPG/RANKL. OCCM-30 cells were cultured with rmIL-33 (5ng/ml,
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20ng/ml) treatment or as vehicle control for 1, 3, 5, 7, 14 days, respectively. Expressions of OPG(A) and RANKL(B) were quantitatively examined by western blot. Data were shown as
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Mean ± SD; n=3, *P<0.05, **P<0.01. (C)The ratios of OPG/RANKL were also calculated and
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analyzed. Data were shown as Mean ± SD; n= 3, *P<0.05, **P<0.01.
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PII:
S0003-9969(19)31184-7
DOI:
https://doi.org/10.1016/j.archoralbio.2020.104663
Reference:
AOB 104663
To appear in:
Archives of Oral Biology
Received Date:
22 November 2019
Revised Date:
4 January 2020
Accepted Date:
9 January 2020
Please cite this article as: Dong X, Feng J, Wen J, Bai D, Xu H, Effect of interleukin-33 on cementoblast-mediated cementum repair during orthodontic tooth movement, Archives of Oral Biology (2020), doi: https://doi.org/10.1016/j.archoralbio.2020.104663
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Effect of interleukin-33 on cementoblast-mediated cementum repair during orthodontic tooth movement
Xiaomeng Dong a, Jie Feng a, Ji Wen a, Ding Baib, Hui Xub,* a State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, Sichuan University, Chengdu, China. b State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases;
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Department of Orthodontics, West China Hospital of Stomatology; Sichuan University, Chengdu, China.
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*Corresponding author: Hui Xu telephone: 86-028-85501474
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e-mail: [email protected]
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address:Department of Orthodontics, West China Hospital of Stomatology, 3rd section of South Renmin Road,
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Chengdu, China, 610041.
Highlights
Heavy orthodontic force increases interleukin-33 expression in periodontal
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tissue.
ST2 is expressed in cementoblast-like cells and is regulated by interleukin-33.
Interleukin-33 abates differentiation and mineralization of OCCM-30 cells.
Interleukin-33
abates
cementogenesis-related
protein
expressions
in
OCCM-30 cells.
Interleukin-33 down-regulates the ratio of osteoprotegerin/RANKL in 1
OCCM-30 cells.
Abstract Objective: This study aims to uncover the role of interleukin-33 on cementoblast-mediated cementum repair. Methods: 6-8-week-old C57BL/6 mice were used to establish the model of orthodontic tooth movement.
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Interleukin-33 and suppression of tumorigenicity2 (ST2) expressions were
immunohistochemically detected in the periodontal tissue. In vitro, cementoblast-like (OCCM-30) cells were cultured in the presence of recombinant mouse interleukin-33 protein (rmIL-33) at a
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1-14 d time frame. ST2 expressions were immunofluorescently labeled and quantitatively
examined. The effects of interleukin-33 on cementoblast differentiation, mineralization and
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proliferation were examined by alkaline phosphatase, alizarin red staining and cell counting kit-8, respectively. To further clarify the effect of interleukin-33 on cementogenesis-related protein
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expressions, runt-related transcription factor 2 (RUNX2), osterix, osteopontin, bone sialoprotein(BSP), osteocalcin, osteoprotegerin (OPG) and receptor activator of NF-КB ligand
Results:
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(RANKL) expressions were examined by western blot.
Orthodontic load of high magnitude induces external apical root resorption, and increases
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interleukin-33 expression in the periodontal tissue of mice. Cells in the cementum express ST2. Interleukin-33 initially down-regulates but later recovers ST2 mRNA and protein levels in
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OCCM-30 cells. Interleukin-33 abates cementoblast differentiation and mineralization, and suppresses RUNX2, osterix, BSP and osteopontin expressions in OCCM-30 cells at the later stage of the culture period. Interleukin-33 enhances RANKL expression, and reduces the ratio of OPG/RANKL in OCCM-30 cells. Conclusion: Orthodontic load of high magnitude induces interleukin-33 expression in the periodontal tissue. Interleukin-33 has a negative effect on cementogenesis via suppressing cementoblast
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differentiation, mineralization and cementogenesis-related protein expressions.
Keywords: interleukin-33, cementoblast, cementum, tooth movement
Introduction External apical root resorption, characterized as a pathological process that brings about resorption of the superficial root cementum, is a frequent side effect secondary to orthodontic treatment (Feller, Khammissa, Thomadakis, Fourie, & Lemmer, 2016; Fernandes et al., 2019;
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Iglesias-Linares & Hartsfield, 2017; Nieto-Nieto, Solano, & Yanez-Vico, 2017). Teeth may also suffer external apical root resorption in the absence of orthodontic treatment, in response to
traumatic, inflammatory, autoimmune or infectious stimuli (Nieto-Nieto et al., 2017). External apical root resorption had been reported to occur in over 90% of orthodontic patients, among
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which, aggressive external apical root resorption (defined as a loss of length exceeding 5 mm) has been seen in 2–5% of orthodontic patients (Feller et al., 2016; Fernandes et al., 2019;
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Iglesias-Linares & Hartsfield, 2017; Nieto-Nieto et al., 2017). However, loss of root length is clinically insignificant in the majority of cases, owing to root remodeling and repair occurring at
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the cellular cementum (Feller et al., 2016).
Dental root remodeling is a dynamic process during which cementum resorption and subsequent
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repair by cellular cementum take place (Feller et al., 2016; Iglesias-Linares & Hartsfield, 2017).Biologically, the susceptibility of the dental root to pathologic resorption is dictated by the
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counterbalance between two key cellular aspects, one is formation and activation of cementoclasts, the other is cementoblast-mediated remineralization and cementogenesis which account for repair
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capabilities (Feller et al., 2016; Iglesias-Linares & Hartsfield, 2017). Signals orchestrating these cellular aspects have the potential to influence the extent and frequency of external apical root resorption, but the molecular and cellular mechanisms which mediate this process are poorly understood. External apical root resorption results from multifactorial causes including intrinsic factors, environmental factors and variables related to treatment (Al-Qawasmi et al., 2003; Nieto-Nieto et al., 2017). Previous studies emphasized intrinsic causes as important etiological factors by 3
reporting that genetic predisposition accounted for at least 50% of variation in post-orthodontic external apical root resorption, with variations in the interleukin-1B gene determining 15% of the differences (Hartsfield, Everett, & Al-Qawasmi, 2004), though it is still unclear how interleukin-1 gene determines the predisposition of root resorption. Interleukin-33 (IL-33) was identified as a member of theinterleukin-1 family and a ligand for the receptor suppression of tumorigenicity2 (ST2) (Liew, Pitman, & McInnes, 2010; Schmitz et al., 2005). IL-33 is constitutively expressed in structural and lining cells, and is released into the extracellular space upon tissue damage and/or mechanical injury (Xu, Turnquist, Hoffman, &
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Billiar, 2017). IL-33 is recognized to act as an alarmin to sense damage and alert neighboring cells following infection or trauma, and has potent influence on a broad range of diseases by targeting various cell types (Kurowska-Stolarska, Hueber, Stolarski, & McInnes, 2011; Palmer & Gabay,
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2011; H. Xu et al., 2017; Xu et al., 2018; J. Xu et al., 2017). The role of IL-33 on cementogenesis and dental root remodeling is unexplored. This study aims to investigate the role of IL-33 in
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cementoblast-mediated cementum repair during orthodontic tooth movement.
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Materials and Methods Animals
All protocols in this study were approved by the Bioethics Committee of Sichuan University. 10
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male C57BL/6 mice (Dashuo, Sichuan, China) were obtained and bred in a temperature-controlled room with a 12/12-hour light–dark cycle. All mice were used at the age of 6–8 weeks.
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Orthodontic tooth movement model
We used a split-mouth method for the orthodontic tooth movement model. After the mice were
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anesthetized by an intraperitoneal injection of 10% chloral hydrate (0.3mg/kg), an closed-coil spring (Mingxin, Chengdu, China) was activated and fixed between the maxillary first molar and maxillary incisor on the right side, to exert a force of 40 g initially without re-activation or interruption during 14 days. The magnitude of force was chosen at a high level to mimic the condition when heavy orthodontic load was applied. (Wolf et al., 2018). The contralateral teeth on the left side were also ligated to the appliance but without activation of the coil spring, serving as vehicle control. The teeth of mice were subjected to orthodontic load or to unloaded control for 14 4
days. Mice were euthanized by an overdose of CO2 inhalation at 14 d. The maxillas were removed and fixed in 4% paraformaldehyde for 48h at 4℃, then were decalcified in 10% disodium ethylenediaminetetraaceticacid (EDTA, pH 7.4) solution for 1 month. After decalcification, the maxillary tissues were dehydrated and embedded in paraffin. 4-μm-thicksections were prepared for experiments.
Immunohistochemistry and Hematoxylin and eosin (HE) staining.
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The tissue sections were dewaxed, rehydrated, then incubated with 0.1% trypsin for 30min at 37℃ for antigen retrieval. The sections were stained with Hematoxylin and eosin for observation of histological changes. Immunohistochemical staining was performed using a mouse/rabbit
streptomyces oval-biotin assay (Zhongshan Golden Bridge Biotechnology, Beijing, China). Briefly,
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the sections were blocked with 3% hydrogen peroxide for 40 min, blocked with 1% BSA for
30min and then incubated with anti-IL33 (1:600, Abcam, USA) or anti-ST2 primary antibodies
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(1:600, Abcam, USA) overnight at 4℃. After being incubated with biotin-labeled goat anti-mouse/rabbit IgG polymer for 10 min and then with horseradish-labeled streptomycin for
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10min at room temperature, the reaction products were visualized with a diaminobenzidine substrate kit (Zhongshan Golden Bridge Biotechnology, Beijing, China). The sections were
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counterstained with haematoxylin or methyl green (Solarbio, Beijing, China). Cell culture and drug administration
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Cementoblast-like (OCCM-30) cells, a murine cell line, were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China) and cultured in alpha-Minimum Essential
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Medium (α-MEM) (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% streptomycin/penicillin (Gibco, USA) as described previously(Oka et al., 2007).After reaching 80% confluence, cell growth was arrested by incubating in serum-free medium for 24 hours. Afterwards, the quiescent cells were cultured in α-MEM with 5% FBS. For drug administration, recombinant mouse interleukin-33 (rmIL-33, R&D, USA) was added to the culture medium (α-MEM plus 5% FBS), at a concentration of 5ng/ml or 20ng/ml. For vehicle control, the culture medium was supplied with phosphate-buffered saline (PBS), instead of recombinant protein. 5
The medium was replaced with fresh medium every other day, to maintain a relatively stable drug concentration during a long culture period. The cultures were divided into five groups. OCCM-30 cells in each group were subjected to rmIL-33 stimulation or vehicle control for 1,3,5,7,14days, respectively. Cells were harvested at indicated time point during the 1-14 d time frame. Experimental procedures were repeated no less than three times for each experimental condition.
Immunofluorescence OCCM-30 cells were seeded onto 4-well glass slides at a density of 4x10^3 cells/well (Millipore,
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USA) for Immunofluorescent labeling of ST2 expression. After being fixed with 4% paraformaldehyde for 20min, the cells were permeabilized with 0.1% triton for 8min and then
blocked with 1% BSA (Biofroxx, Germany) for 30min. The cells were incubated with anti-ST2 primary antibody (1:200, Abcam, USA) for 16h at 4℃, and then incubated with goat anti-rabbit
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IgG antibody (1:50, Bioss, China) for 1 h at 37℃.The nuclei were stained with
Cell Counting Kit-8 (CCK-8)
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fluorescence microscope (Leica, USA).
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4,6-diamidino-2-phenylindole(DAPI) (solarbio, Beijing, China).Images were captured by a
OCCM-30 cells were seeded into 96-wellplates at a density of 4x10^3 cells/well. After being
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cultured overnight, the cells were treated with rmIL-33 (5ng/ml, 20ng/ml) or vehicle control for 1,3,5,7 days, respectively. The cells were washed with PBS, then incubated with culture medium supplemented with 10% CCK-8 (Dojindo, Japan) for 30 min at 37℃. The optical density (OD)
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values were read at 450nm wavelength.
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Alkaline phosphatase (ALP) staining and quantitative analysis OCCM-30 cells were seeded into 12-well plates at a density of 5x10^4cells/well. After being cultured overnight, the cells were treated with rmIL-33 (5ng/ml, 20ng/ml)or vehicle control for 7 days. Cells were stained using BCIP/NBT alkaline phosphatase color development kit (Beyotime, Beijing, China). ALP activity was quantified by alkaline phosphatase assay kit (Beyotime, Beijing , China).
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Alizarin red staining (ARS) and quantitative analysis OCCM-30 cells were seeded into 12-well plates at a density of 5x10^4 cells/well. After being cultured overnight, the cells were cultured in osteogenic differentiation medium, that is supplemented with10^8mmol/L dexamethasone (Sigma, USA), 0.2mmol/L ascorbic acid (J&K, Beijing, China) and10mmol/L β-glycerophosphate (Sigma, USA), with or without rmIL-33 (0ng/ml, 5ng/ml, 20ng/ml) for 21days. After being washed with PBS, the cells were incubated with 0.1% alizarin red solution for 10 min (PH=4.3, Solarbio, Beijing, China). After being captured by a camera, the mineralized nodules were dissolved in 2ml 10% cetylpyridinium
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chloride (J&K, Beijing, China), then the OD values were read at 562nm wavelength.
Quantitative Real-time PCR (qRT-PCR)
OCCM-30 cells were seeded into 6-well plates at a density of 20x10^4 cells/well, and treated with
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rmIL-33 (5ng/ml, 20ng/ml) or vehicle control for 1,3,5,7,14 days, respectively. The total RNAs were extracted with trizol reagent (Thermo, USA). The 1μgcomplementary DNA (CDNA)
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synthesis was perfomed using PrimeScript™ RT reagent Kit (TaKaRa, Japan). The premier sequence:ST2 (17082):5’-GCCACAGGACATCAGCCAAGAAG-3’ (forward) and 5’-GA-
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CGGCCACCAGATCATTCACAG-3’ (reverse); GAPDH (14433):5’-AGGTCGGTGTGAACGGATTTG-3’ (forward) and 5’-TGTAGACCATGTAGTTGAGGTCA-3’ (reverse).QRT-PCR was
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performed using TB Green® Fast qPCR Mix (TaKaRa, Japan) in Quant Studio™3 Flex Real-time PCR System. The ST2 relative mRNA expression was calculated using the 2 ^-ΔΔCt method.
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Western blot assay
OCCM-30 cells were seeded into 6-well plates at a density of 20x10^4 cells/well, and treated with
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rmIL-33 (5ng/ml, 20ng/ml) or vehicle control for 1, 3, 5, 7, 14 days, respectively. Proteins were extracted with a protein extraction kit (SAB, USA). The protein concentration were measured using a BCA protein assay kit (Beyotime, Bijing, China). Equal amounts of protein extracts were separated on 10% SDS–polyacrylamide gels (Biorad, USA), and then transferred onto polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked in 5% skim milk (Biofroxx, Germany) for 1h at room temperature, then incubated with rabbit anti-runt-related transcription factor 2 (RUNX2) (1:1000,Huaan, Shanghai, China), rabbit anti-osteopontin 7
(1:1000,Proteintech, USA), rabbit anti-osterix (1:1000, Abcam, USA), mouse anti-bone sialoprotein (BSP) (1:300, Santa Cruz, USA), rabbit anti-osteocalcin
(1:1000, Affinity, USA),
rabbit anti-osteoprotegerin (OPG) (1:1000, Affinity, USA), rabbit anti-receptor activator of NF-КB ligand (RANKL) (1:1000, Affinity, USA), for 16h at 4℃. After being washed with TBST, the membranes were incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG (SAB, USA) and visualized with chemiluminescent substrate (US Everbright lnc, USA). Quantitation of the protein levels were analyzed by Image J software (National Institutes of Health,
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Bethesda, MD).
Statistical analysis
All data were analyzed with Graphpad Prism 7.0 (Graphpad Software Inc, San Diego, CA).
Experimental results were presented as mean ± SD of triplicate determinations. Unpaired t-test
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was used for comparison of two groups. Analysis of variance (ANOVA) were used for comparison among groups, and Tukey's multiple comparison test were used for multiple comparisons between
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pairs. A probability at the 5% level or less (P < 0.05) was considered statistically significant.
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Results
Orthodontic force of high magnitude leads to increased IL-33 expressions in the periodontal
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tissue and induces root resorption in mice
We examined interleukin-33 and ST2 expressions in the periodontal tissue under orthodontic load
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(figure 1). Immunohistochemistry staining revealed increased IL-33 expression in compression areas of the PDL under 40 g orthodontic force, compared with unloaded control (figure 1A).
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Immunostaining for ST2 was positive in the periodontal tissue and in the dental pulp of mice, irrespective of orthodontic loading or unloaded control (figure 1B). Interestingly, ST2 expression was observed in cells in the cementum (figure 1B). It is well recognized that orthodontic forces of high magnitude cause root resorption, and we confirmed this by finding resorption lacunae in compression areas of the root surface under 40 g orthodontic force in a mouse model (figure 1B, C). The concomitant occurrence and adjacent location of root resorption and IL-33/ST2 expression lead us to investigate the role of IL-33 in cementum remodeling. 8
Cementoblast-like (OCCM-30) cells express ST2. IL-33 initially down-regulates but later recovers ST2 mRNA and protein levels in OCCM-30 cells. Immunofluorescence showed ST2 expression in OCCM-30cells (figure 2A). We also examined gene and protein levels of ST2 expression by qRT-PCR (figure 2B) and western blot (figure 2C). To test whether ST2 expression is regulated by its ligant, OCCM-30 cells were cultured in the presence of different concentrations of rmIL-33 protein. The expression levels of ST2 were quantitatively examined at 1-14d time frame during the culture period. ST2 mRNA levels in OCCM-30 cells were down-regulated at 3-7d (figure 2B, P<0.05), and recovered at 14 d in the
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presence of 5ng/ml or 20ng/ml rmIL-33. Similar trend was found for the change of ST2 protein
levels over time in the presence of rmIL-33 stimulation, with a significant up-regulation of ST2 by 20ng/ml rmIL-33at 14 d (figure 2C, P<0.05).
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IL-33 abates cementoblast differentiation and mineralization of OCCM-30 cells
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We investigated the effects of IL-33 on OCCM-30 cell differentiation by ALP staining and quantitative analysis (figure 3A). ARS and quantitative analysis were performed to detect the
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effect of interleukin-33on cell mineralization (figure 3B). rmIL-33 inhibited cementoblast differentiation of OCCM-30 cells at a concentration of 5ng/ml or 20ng/ml (figure 3A, P<0.05). Formation of calcium nodules were observed in OCCM-30 cell cultures at 21d. Treatment of
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5ng/ml or 20ng/ml rmIL-33 attenuated OCCM-30 cell mineralization, compared with vehicle control (figure 3B, P<0.05).
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The proliferative response of cementoblast-like cells to IL-33 was examined by a CCK-8 assay kit. As showed in figure 3C, 5ng/ml rmIL-33 had no effect on OCCM-30 cells proliferation at 1-7d
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time frame during the culture period, while 20ng/ml rmIL-33 didn’t significantly inhibited cell proliferation until 7 d (figure 3A, P<0.05). IL-33 suppresses expressions of RUNX2, osterix, BSP and osteopontin in OCCM-30 cells at the later stage of the culture period To further clarify the effect of IL-33 on cementogenesis-related protein expressions in OCCM-30
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cells, expressions of RUNX2, osterix, osteopontin, osteocalcin and BSP in OCCM-30 cells were examined by western blot (figure 4-5) at 1-14 d time frame under constant rmIL-33 stimulation. As presented in figure 4, administration of 5ng/ml rmIL-33 has no significant effect on expressions of RUNX2 and osteopontin at 1-7 d, but inhibited their expressions at 14 d (P<0.05). rmIL-33 inhibited osterix expressions as early as 3-5 d at a concentration of 20ng/ml (figure 4, P<0.05). rmIL-33 inhibited BSP expressions at 3 d, with a trend towards abating BSP levels at 14 d (figure 5A). 5ng/ml or 20ng/ml rmIL-33 didn’t significantly affect osteocalcin expressions in OCCM-30 cells at 1-14 d (figure 5B, P>0.05).
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IL-33 promotes RANKL expressions in OCCM-30 cells, and down-regulates the ratio of OPG/RANKL.
We investigated expressions of OPG and RANKL proteins in OCCM-30 cells over time under
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IL-33 stimulation, and calculated the ratio of OPG/RANKL. The time course analysis revealed a
trend towards initial suppression but later promotion of OPG expressions in OCCM-30 cells under
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rmIL-33 stimulation at a concentration of 5ng/ml or 20ng/ml (figure 6). In contrast, the change of RANKL expression showed a trend towards initial up-regulation, then down-regulation, followed
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by a significant increase at 14 d under rmIL-33 administration (figure 6). IL-33 down-regulated the ratio of OPG/RANKL in OCCM-30 cells at the 1-14 d time frame during the culture period,
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Discussion
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compared with vehicle controls (figure 6, P<0.05).
Orthodontic load-induced external apical root resorption was described as a complex, sterile
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inflammatory process that involves a dynamic interplay between local immune microenvironment and dental hard tissue(Feller et al., 2016). Inflammatory cytokines orchestrate this signaling network and influence the process of root resorption. Polymorphism of genes encoding cytokines and growth factors constitute an intrinsic factor in the pathogenesis of orthodontic load-induced external apical root resorption (Feller et al., 2016; Nieto-Nieto et al., 2017). Research groups have analyzed the genetic influence on the susceptibility to post-orthodontic external apical root resorption, such as interleukin-1 gene cluster, tumor necrosis factor-α, OPG, RANK and
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osteopontin gene, among which,interleukin-1 gene was highlighted by most studies(Al-Qawasmi et al., 2003; Bastos Lages et al., 2009; Gulden et al., 2009; Iglesias-Linares et al., 2012; Nieto-Nieto et al., 2017). The relationship between interleukin-1A or interleukin-1B gene polymorphisms and the risk of post-orthodontic external apical root resorption was determined (Bastos Lages et al., 2009; Gulden et al., 2009; Iglesias-Linares et al., 2012). Persons homozygous for allele 1 of the interleukin-1B gene have a 5.6-fold increased risk of post-orthodontic external apical root resorption compared with heterozygous subjects and those homozygous for allele 2 (Al-Qawasmi et al., 2003). Researchers tried to explain the mechanism by speculating that allele 1
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of interleukin-1B gene may cause reduced levels of interleukin-1β protein and impaired alveolar bone resorption, and thus result in prolonged stress concentrated around the adjacent
tooth root, without clarifying whether and how these genes have direct influence on the cementum
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(Hartsfield et al., 2004).
Root resorption is counterbalanced by cementum repair, otherwise would cause permanent loss of
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root length (Feller et al., 2016). Cementoblast-mediated mineralization and cementogenesis play vital roles in cellular cementum repair (Feller et al., 2016; Rego et al., 2011). Factors regulating
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cementogenesis determine the repair capabilities of the root, thus influencing the extent and frequency of external apical root resorption(Feller et al., 2016; Iglesias-Linares & Hartsfield, 2017). Previous studies demonstrated the association of root resorption with various cytokines
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including interleukin-1α, interleukin-1β,tumor necrosis factor-α and interleukin-6, though didn’t look into the mechanism whether and how these cytokines affect cementogenesis and root
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repair(Bastos et al., 2017; Kikuta, Yamaguchi, Shimizu, Yoshino, & Kasai, 2015; Kunii et al., 2013; Matsumoto, Sringkarnboriboon, & Ono, 2017; D. Zhang, Goetz, Braumann, Bourauel, &
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Jaeger, 2003). Limited evidence was from Diercke (Diercke, Konig, Kohl, Lux, & Erber, 2012), who presented that combination of compression and interleukin-1β stimulation impeded bone sialoprotein and cementum Protein 1 expression in human primary cementoblasts, while compression or interleukin-1β stimulation alone led to increased levels of bone sialoprotein and decreased levels of cementum Protein 1. In this study, we revealed IL-33, a member of the interleukin-1 family, as a negative regulator of cementogenesis via suppressing cementoblast differentiation and mineralization and cementogenesis-related protein expressions. IL-33 may 11
mediate impairment of cementum repair and consequently aggravate root resorption. IL-33 is an immunoregulatory cytokine which plays diverse and context-specific roles in various diseases (Kurowska-Stolarska et al., 2011; Palmer & Gabay, 2011; H. Xu et al., 2017). Recently, IL-33 was demonstrated to have interaction with skeletal system, though its role in osteogenesis remains elusive (Heckt et al., 2016; Kukolj et al., 2019; Mine, Makihira, Yamaguchi, Tanaka, & Nikawa, 2014; Saidi et al., 2011; Saleh et al., 2011). Cementum is similar to bone in many aspects, while cementum differs from bone in cellular components and having limited capacity for remodeling (Feller et al., 2016). It is controversial whether osteoblasts and cementoblasts are
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derived from a common precursor cell, though they both share many properties with respect to
phenotype, function and driven factors (Cao et al., 2015; Cao et al., 2012; Feller et al., 2016). It
was reported that IL-33 increases mRNA expression and secretion of RANKL in MC3T3-E1 cells
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(Mine et al., 2014), while induces expression of Tnfsf11 (RANKL-encoding gene)
in primary osteoblasts without soluble RANKL release (Heckt et al., 2016). Consistently, we
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showed increased expression of RANKL protein in OCCM-30 cells under IL-33 stimulation. Saleh(Saleh et al., 2011) demonstrated that IL-33 promoted matrix mineral deposition in primary
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osteoblasts in vitro. In contrast, we showed suppressed mineralization of OCCM-30 cells after IL-33 administration. These comparisons indicated that IL-33-driven responses are delicately
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cell-type-specific.
The IL-33 receptor ST2 is a member of the interleukin-1 receptor family (Kurowska-Stolarska et al., 2011; Palmer & Gabay, 2011; H. Xu et al., 2017). Membrane-bound ST2 is the functional
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component for IL-33 signaling (Kurowska-Stolarska et al., 2011; Palmer & Gabay, 2011; H. Xu et al., 2017). Observed ST2 expression in cells in the cementum and OCCM-30 cells indicated them
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as target cells of IL-33. Interestingly, we found that ST2 levels was initially down-regulated but later recovered by IL-33 in vitro, and in vivo, heavy orthodontic forces induced increased IL-33 expression with basal-level expression of ST2 in the periodontal tissue at corresponding time point, compared with unloaded control. It can be speculated that a negative feedback loop may exist between IL-33 and its receptor at early but not the later stage of culture period. Continuous IL-33 stimulation eliminated the negative feedback, and even drives a positive feedback at a high concentration, as we saw that 20ng/ml rmIL-33significantly up-regulated ST2 expression at 14 d 12
in vitro. Notably, the time frame when ST2 expression was recovered corresponds with that when cementoblast differentiation and cementogenesis-related protein expressions were suppressed. The regulation of ST2 expression by IL-33 may help explain the time-dependent change of effect of IL-33. Under IL-33 stimulation, the IL-33/ST2 signaling may be initially cancelled out by the negative feedback, and exert its power over time as the negative feedback disappeared. In the process of cementum repair, osteopontin and BSP are noncollagenous matrix proteins playing vital roles in formation of cementoid repair matrix at early stage of root repair (Feller et al., 2016; Jager et al., 2008). Osteopontin and osteocalcin contribute to the recruitment of
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cementoblast precursors to the root surface and to their subsequent adhesion, proliferation and differentiation(Feller et al., 2016; Jager et al., 2008). Mineralization ensues with cementoblast
proliferation and differentiation, forming reparative cementum and promoting cementogenesis
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(Feller et al., 2016). Working upstreams are the transcription factors RUNX2and osterix. RUNX2
is the master transcription factor that positively regulates the genes encoding alkaline phosphatase,
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collagen type 1,osteocalcin, and osterix (P. Zhang, Wu, Jiang, Jiang, & Fang, 2012). Osterix is a transcription factor functions molecularly downstream from RUNX2, and upregulates osteopontin,
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osteocalcin, and bone sialoproteingene and protein levels(Cao et al., 2015; Hakki et al., 2010). Osterixis essential for the differentiation of precementoblasts into mature cementoblasts (Cao et al., 2015; Feller et al., 2016). We investigated these cementogenesis-related protein expressions in
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cementoblast-like cells and found they were suppressed by interleukin-33 at not the early but the later stage of culture period, indicating that continuous IL-33 stimulation may cause impaired
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cementum repair.
Cautions should be taken against over-interpretation of the in-vitro effects on cementoblast-like
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cells, as biological properties of a cementoblast-like cell line is not identical to that of primary cementoblast culture. It is also worth noting that the in-vitro experiments cannot reproduce the multitude of factors in vivo. The in-vivo effects are derived from interactions among different types of cells and the extracellular matrix in the periodontal tissue. Further investigations are needed to explore the mechanism by which IL-33 suppresses differentiation and mineralization of cementoblasts, and to examine whether IL-33 has effects on other types of cells in the periodontal tissue. 13
In general, we revealed that orthodontic forces of high magnitude caused external apical root resorption in mice, and concomitantly induced increased IL-33 expression in the periodontal tissue. The IL-33 receptor ST2 was found expressed in the cementum and in cementoblast-like (OCCM-30) cells. IL-33 abated cementoblast differentiation and mineralization of OCCM-30 cells, inhibited expressions of RUNX2, osterix, osteopontin and BSP in these cells at the later stage of the culture period. IL-33 promoted RANKL expressions in OCCM-30 cells, and down-regulated the ratio of OPG/RANKL. These results indicated an anti-reparative effect of IL-33 on cementum remodeling under orthodontic load, mediated by suppressed cementoblast
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differentiation and mineralization and down-regulation of cementogenesis-related proteins.
Declaration of Competing Interest
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The authors declare no conflict of interest.
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Funding
This study was funded by National Nature Science Foundation of China (Grant Nos 81701006 and
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81870804).
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Conflict of interest
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The authors declare no conflict of interest.
Author Contributions: The conception and design of the study：Hui Xu, Xiaomeng Dong, Ding Bai Acquisition of data：Xiaomeng Dong, Jie Feng Analysis and interpretation of data: Xiaomeng Dong , Ji Wen Drafting the article: Xiaomeng Dong, Hui Xu 14
Revising it critically for important intellectual content: Hui Xu, Ding Bai, Jie Feng, Ji Wen, Final approval of the version to be submitted: Xiaomeng Dong, Jie Feng, Ji Wen, Ding Bai, Hui Xu
Acknowledgement The authors would like to acknowledge Bin Li for helping review and edit this manuscript, and
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acknowledge Chaoran Xue and Mengting He for data analysis.
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29(6), 1083-1089. doi:10.3892/ijmm.2012.934
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Figure Captions Figure 1. Heavy orthodontic force increases interleukin-33 expressions in the periodontal tissue and induces root resorption in mice. Representative images showing the expressions of interleukin-33 (A) and ST2 (B) in the periodontal tissue at 14 d under 40 g orthodontic force or unloaded control. The study areas were all selected around the distal roots of the maxillary first molars. →: the direction of tooth movement; scale bar: 100μm (upper panel), 50μm (lower panel). (C) Representative HE staining images of the periodontal tissue at 14 d under orthodontic load or
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unloaded control. The study areas were all selected around the distal roots of the maxillary first molars. →: the direction of tooth movement; scale bar: 200 μm (upper panel), 50 μm
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(lower panel).
Figure 2. Cementoblast-like (OCCM-30) cells express ST2. ST2expressions were initially down-regulated but later recoveredby interleukin-33 stimulation.(A) Representative 19
immunefluorescence staining for ST2 in cementoblast-like (OCCM-30) cells. Green: ST2, Blue: DAPI. Scale bar: 100μm. OCCM-30 cells were cultured in the presence of rmIL-33(5ng/ml, 20ng/ml) or as vehicle control for 1, 3, 5, 7, 14 days, respectively. Gene and protein levels of ST2 expressionswere quantitatively examinedby qRT-PCR (B) and western blot (C). Data were shown
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as Mean ± SD; (B) n=9 per group,(C) n=3 per group.*P<0.05, **P<0.01.
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Figure 3. Interleukin-33 abates cementoblast differentiation and mineralization of OCCM-30 cells. (A) Representative ALP staining of OCCM-30 cells treated with rmIL-33 (5ng/ml, 20ng/ml) or vehicle control for 7 days. The ALP activity was also quantitatively analyzed. Data were shown as Mean ± SD; n=9.*P<0.05, **P<0.01. (B)Representative ARS of OCCM-30 cells cultured in osteogenic differentiation medium with rmIL-33 (5ng/ml, 20ng/ml) or vehicle control for 21 days. The alizarin red activity was also quantitatively analyzed. Data were shown as Mean ± SD; n=9.*P<0.05, **P<0.01. (C)OCCM-30 cells were treated with rmIL-33 (5ng/ml,
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20ng/ml) or vehicle control for 1, 3, 5, 7 days, respectively. The proliferative activities of these
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cells were examined by CCK-8 assay. Data were shown as Mean ± SD; n=6.*P<0.05, **P<0.01.
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Figure 4. Interleukin-33 suppresses expressions of RUNX2, osterixand osteopontin in OCCM-30 cells at the later stage of the culture period. OCCM-30 cells were cultured with
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rmIL-33 (5ng/ml, 20ng/ml) treatment or as vehicle control for 1, 3, 5, 7, 14 days, respectively. Expressions of RUNX2 (A), osterix (B) and osteopontin (C) were quantitatively examined by western blot.Data were shown as Mean ± SD; n=3-8, *P<0.05, **P<0.01.
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Figure 5. Effect of interleukin-33 on the expressions of BSP and osteocalcin in OCCM-30 cells. OCCM-30 cells were cultured with rmIL-33 (5ng/ml, 20ng/ml) treatment or as vehicle control for 1, 3, 5, 7, 14 days, respectively. Expressions of BSP (A) and osteocalcin(B) were
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**P<0.01.
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quantitatively examined by western blot. Data were shown as Mean ± SD; n=3-6, *P<0.05,
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Figure 6. Interleukin-33 promotes RANKL expressions in OCCM-30 cells, and down-regulates the ratio of OPG/RANKL. OCCM-30 cells were cultured with rmIL-33 (5ng/ml,
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20ng/ml) treatment or as vehicle control for 1, 3, 5, 7, 14 days, respectively. Expressions of OPG(A) and RANKL(B) were quantitatively examined by western blot. Data were shown as
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Mean ± SD; n=3, *P<0.05, **P<0.01. (C)The ratios of OPG/RANKL were also calculated and
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analyzed. Data were shown as Mean ± SD; n= 3, *P<0.05, **P<0.01.
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