Protein and mRNA expressions of IL-6 and its key signaling factors under orthodontic forces in mice: An in-vivo study

Introduction

The purpose of this study was to investigate the mechanical loading-induced changes in protein and mRNA expressions of interleukin-6 (IL-6) and its key signaling factors glycoprotein 130 (gp130), signal transducer and activator of transcription 3 (STAT3), and the Src homology phosphotyrosine phosphatase (SHP2) at the tension and compression sides of the teeth in mouse models.

Methods

A total of 55 C57B/6 mice (10 weeks old) were divided into 3 groups. Orthodontic force was applied in group A (experimental group, n = 30); the tooth movement device was placed without activation in group B (sham control group, n = 15), and group C (blank control group, n = 10). Tooth movement was induced by a nickel-titanium coil spring inserted between the maxillary left incisor and the first molar with a force of approximately 4 g. The animals were killed 12 days after the interventions; protein and mRNA expressions of IL-6, gp130, STAT3, and SHP2 in the periodontal tissues were observed with immunohistochemistry and in-situ hybridization, respectively.

Results

In contrast with the control groups, we observed enhanced expressions of IL-6, gp130, STAT3, and SHP2 protein and mRNA at the mesial and distal sides of the teeth with application of orthodontic forces in the experimental group. In contrast with the distal side, we observed enhanced expression of gp130 protein and mRNA at the mesial side in the experimental group.

Conclusions

We observed enhanced expression of IL-6 and its key signaling factors gp130, STAT3, and SHP2 protein and mRNA at the tension and compression sides of the teeth with application of orthodontic forces. The mechanical loading applied for orthodontic tooth movement might induce changes in protein localization and mRNA expression patterns of IL-6 and its key signaling factors gp130, STAT3, and SHP2 at the tension and compression sides of the periodontal ligaments of the teeth in mouse models. The result might demonstrate the special role of IL-6 and its key signaling factors in the alveolar bone-modeling process.

Highlights

  • There is enhanced expression of IL-6 and its key signaling factors gp130, STAT3, and SHP2 protein and mRNA in periodontal tissues when orthodontic forces were applied.

  • IL-6 and its downstream signaling pathways might be activated during orthodontic treatment.

  • The result might demonstrate a special role of IL-6 and its key signaling factors in alveolar bone modeling process.

Orthodontic tooth movement is achieved when periodontal ligament (PDL) and alveolar bone modeling occur after the appropriate application of mechanical force for tooth movement inducing the periodontal biologic response. The differentiation of osteoclasts and the resorption of bone at the compression side and the osteoblast differentiation and bone formation at the tension side are both involved in the bone-modeling process. This process is composed of series of complicated molecular signal-regulating networks, by means of which the orthodontic force-related signals are transducted into the biochemical signals both extracellularly and intracellularly. Previous researches have confirmed interleukins’ major role when this complex molecular signaling networks functions.

Interleukin-6 (IL-6) is a multifunctional cytokine in diffuse biologic activities, participating in regulating immunity, neuroendocrine function, inflammation, and tumor genesis, with both proinflammatory and anti-inflammatory effects. Moreover, IL-6 was found to be an important regulator in both bone formation and bone resorption in previous in-vivo studies. In-vitro studies have demonstrated that IL-6 could be produced by osteoblasts and fibroblasts in periodontal tissues, inducing bone resorption alone and in concert with other bone-resorbing agents. IL-6 is remarkably expressed during orthodontic force-related alveolar bone modeling and root resorption, and it may be a major mediator in the transduction of orthodontic force signals.

Glycoprotein 130 (gp130) is the central player of the receptor complex formed by IL-6–type cytokines during the activation of the IL-6 signaling pathway, which is composed of 918 amino acids with a single transmembrane domain. Gp130 itself does not seem to have tyrosine kinase activity and cannot bind directly to its ligand IL-6; however, it plays an important role in the formation of high-affinity IL-6 binding sites by associating with the IL-6/IL-6R complex in transduction of the IL-6 signal. The IL-6/IL-6R complex introduces the homodimerization of gp130 and subsequent association with tyrosine kinases. This conducts extracellular IL-6 signals into the cells, activating the downstream signaling pathways and regulating osteoblastic and osteoclastic activities.

Janus kinase (JAK) activation by gp130 results in activation of the signal transducers and activators of transcription (STAT) STAT1 and STAT3 and the SHP2/ras/Mitogen activated protein kinase (MAPK) signaling pathway. STAT3 is a key transcription factor in the JAK-STAT pathway, whereas SHP2 is 1 essential signaling molecule in multiple MAPK pathways. It has been demonstrated that the JAK-STAT pathway mediates signals that could suppress proliferation, promote apoptosis, or promote bone-forming or bone-breaking, whereas the MAPK pathway mediates signals that could promote mitosis, suppress apoptosis, and inhibit bone-forming or bone-breaking. As a key transcription factor in the JAK-STAT signaling pathway, STAT3 can be phosphorylated via the homologous dimerization of the SH2 domain; the activated STAT3 could be transferred into the nucleus to regulate the transcription of the target genes, thus exerting its roles in promoting osteoblastic and osteoclastic activities inside the target cells.

Changes in the expressions of these key proteins are directly related to the transduction of IL-6 signals and the final biologic effects. So far, studies on the effects of the IL-6 signaling pathway on orthodontic tooth movement are limited to the localized expression of IL-6 in the periodontal or dental tissues. However, the signaling pathway downstream of IL-6 in orthodontic treatment has not been fully established in theoretical research. In our study, we tried to elucidate that the mechanical loading applied for orthodontic tooth movement might induce changes in protein localization and mRNA expression patterns of IL-6 and its key signaling factors gp130, STAT3, and SHP2 at the tension and compression sides of the PDL of the teeth in mice.

Material and methods

Fifty-five 10-week-old male C57B/6 mice were purchased from Sichuan University laboratory animal center (Sichuan, China) and givensolid food and tap water. During the experiment, the mice were kept in cages in a room maintained at 25°C with a 12 to 24 hour light and dark cycle. All animal care and experimental procedures were performed according to the guidelines for animal experimentation of Sichuan University. All animal experiments were approved by the animal welfare committee of Sichuan University.

The mice were divided into 3 groups. Orthodontic force was applied in group A (experimental group, n = 30); the tooth movement device was placed without activation in group B (sham control group, n = 15) and group C (blank control group, n = 10).

The mice in groups A and B were anesthetized with an intraperitoneal injection of 10% chloral hydrate (0.35 ml/100 g), and then they were fixed in a supine position and given a mouth gag. In group A, the nickel-titanium coil spring (Ming Xing Spring, Chengdu, China), of which the force level of the spring was set to approximately 4 g with an electronic force gauge (Yueqing Handpi Instruments, Zhejiang, China), was inserted between the maxillary left incisors and the first molar fixed with 0.1-mm stainless steel wires, moving the maxillary left first molar in the mesial direction. To prevent the detachment of the appliance from the interproximal contact between the maxillary incisors, the anterior end of the spring was reinforced with dental adhesive resin (3M, St Paul, Minn) ( Fig 1 ).

Fig 1
C57B/6 mouse model of orthodontic tooth movement.

The mice were killed with an overdose of anesthetic 12 days after the interventions. Maxillary segments were dissected, rinsed, and immersed in 4% paraformaldehyde solution containing diethyl pyrocarbonate for fixation. After decalcification in 10% edathamil (pH: 7.4) at 4°C for 3 weeks, the specimens were dehydrated in a graded ethanol series and then fixed in paraffin. Mesial and distal serial 4-μm sections were cut along the longitudinal axis of the molar and then mounted on polylysine-coated glass slides.

For the detection of the protein expressions, the paraffin-embedded sections were dewaxed with xylene and dehydrated with a graded series of ethanol. Endogenous peroxidase activity was blocked and inactivated with 3% hydrogen peroxide. Immunohistochemistry of IL-6, gp130, STAT3, and SHP2 was performed according to the manual of the immunohistochemical assay kit (Zhongshan Jinqiao Biological Technology, Beijing, China), with phosphate-buffered saline solution replacing the primary antibody in group C. The sections were developed with diaminobenzidine tetrahydrochloride, counterstained with hematoxylin, dehydrated with a graded series of ethanol, and made transparent with xylene. Finally, the sections were mounted with neutral balsam and observed under a light microscope. Rabbit antimouse IL-6, gp130, STAT3, and SHP2 polyclonal antibodies (Abcam, Cambridge, United Kingdom) were used during the experiment.

For the detection of the mRNA expressions, the sections were dewaxed and rehydrated sequentially with xylene and gradient ethanol, and treated with 3% hydrogen peroxide at room temperature for 10 minutes, followed by flushing with distilled water (5 minutes each). Pepsin freshly diluted with 3% citric acid was used to digest the sections at 37°C for 30 minutes. According to the manual of the in-situ hybridization kit (Boster Biological Engineering, Wuhan, China), the in-situ hybridization was performed for the mRNA of IL-6, gp130, STAT3, and SHP2. The sections were developed with diaminobenzidine tetrahydrochloride, counterstained with hematoxylin, dehydrated through a graded series of ethanol, and made transparent with xylene. Finally, the sections were mounted with neutral balsam and observed under a light microscope. The oligonucleotide sequences for in-situ hybridization probes were designed by BLAST sequence alignment (Songyue Biological Technology, Nanjing, China) as follows:

  • IL-6: 5′-CTCCGCAAGAGACTTCCAGCCAGTTGCCTT-3′

  • gp130: 5′-TACAACGTATCACTACTCAGAGAGGACA-3′

  • STAT3: 5′-CTTGGTGCAGAGGAGCAATGGGGAGTGTAA-3′

  • SHP2: 5′-TGTCCTTCTTTTCGGCTATAGA-3′

The inverted microscope (IX71; Olympus, Tokyo, Japan) was applied for immunohistochemical staining and sectioning and for observing the in-situ hybridization sections. Two visual fields (400 times magnification) were selected at the mesial and distal sides of the tooth roots randomly, and images were collected with a cell image analysis system (ACT-1; Nikon, Tokyo, Japan).

Results

For the protein expressions of IL-6, gp130, STAT3, and SHP2, immunohistochemistry staining showed that intensities of positive expressions of IL-6, gp130, STAT3, and SHP2 protein in the periodontal tissues at the mesial and distal sides were significantly enhanced in group A compared with groups B and C ( Figs 2-5 ). In group A, enhanced expressions of IL-6, gp130, STAT3, and SHP2 protein in the periodontal extracellular matrix and in the cytoplasm of fibroblasts, osteoblasts were observed. The positive expression of gp130 protein at the mesial side was stronger than at the distal side in group A ( Fig 3 ).

Fig 2
IL-6 immunohistochemical staining (400 times magnification): intensities of positive expressions of IL-6 in periodontal tissues at mesial and distal sides were significantly enhanced in group A compared with groups B and C. A1 , Group A, tension side; A2 , group A, compression side; B1 , group B, distal side; B2, group B, mesial side; C1 , group C, distal side; and C2 , group C, mesial side. r , root; b , alveolar bone; red arrow , osteoblast; yellow arrow , fibroblast.

Fig 3
gp130 immunohistochemical staining (400 times magnification): intensities of positive expressions of gp130 in periodontal tissues at mesial and distal sides were significantly enhanced in group A compared with groups B and C, in the same sequences as in Figure 2 .

Fig 4
STAT3 immunohistochemical staining (400 times magnification): intensities of positive expressions of STAT3 in periodontal tissues at mesial and distal sides were significantly enhanced in group A compared with groups B and C, in the same sequences as in Figure 2 .

Fig 5
SHP2 immunohistochemical staining (400 times magnification): intensities of positive expressions of SHP2 in periodontal tissues at mesial and distal sides were significantly enhanced in group A compared with groups B and C, in the same sequence as in Figure 2 .

For the mRNA expressions of IL-6, gp130, STAT3, and SHP2, in-situ hybridization showed that intensities of positive mRNA expressions of IL-6, gp130, STAT3, and SHP2 mRNA in the periodontal tissues at the mesial and distal sides were significantly enhanced in group A compared with groups B and C ( Figs 6-9 ). In group A, the IL-6, gp130, STAT3, and SHP2 mRNA expressions in osteoblasts and periodontal membrane fibroblasts in the periodontal tissues were significantly increased with regard to the number of positive cells and staining intensity. The positive expression of gp130 mRNA at the mesial side was stronger than at the distal side in group A ( Fig 7 ).

Dec 12, 2018 | Posted by in Orthodontics | Comments Off on Protein and mRNA expressions of IL-6 and its key signaling factors under orthodontic forces in mice: An in-vivo study
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