Assessment of the RANKL/RANK/OPG axis and a ratio between its components in bone tissue of patients
As the RANKL/RANK/OPG signaling axis regulates the interplay between osteoclasts and osteoblasts that can influence bone remodeling [6], we first determined the content of the RANKL/RANK/OPG system components in the bone tissue of patients with tooth impaction. The results of the study presented in Fig. 1 show that there is no difference in the relative protein content of RANK (Fig. 1 A, B, Tukey's test, p-value >0.05) and osteocalcin (Fig. 1 A, D, Tukey's test, p-value >0.05) between the samples of healthy persons (control group) and samples, that were taken near the normally erupted third molar in patients with tooth impaction. At the same time, it was shown a slight tendency to decreased content of osteoprotegerin (by 1.2 times) in the bone tissue near the healthy tooth in patients with tooth impaction compared with the control (Fig. 1C, Tukey's test, p-value 0.048) and we observed a significant decrease in the RANKL content (membrane-bound form – by 1.6 folds, Tukey's test, p-value 0.021, and soluble form – by 10 folds, Tukey's test, p-value 0.003) (Fig. 2C, 2D).
Fig. 1 Immunoblotting analysis of RANK, OPG, and osteocalcin in bone tissue samples: representative immunoblots are shown (A) and quantified using β-actin as a loading control. The bar graphs of RANK (B), OPG (C) and osteocalcin (D) are presented as arithmetic means ± SEM (n = 6/group): 1 – bone tissue of healthy persons (control group); 2 – bone tissue, that was taken near the healthy tooth in patients with tooth impaction; 3 – bone tissue, collected near the impacted tooth. Experiments were performed in triplicate and *p<0.05 denotes significance compared with control, #p<0.05 denotes significance compared with bone tissue taken near the healthy tooth in patients with tooth impaction.
Completely different results were obtained when studying the area of the impacted third molars. Western blot analysis data revealed a 1.73-fold elevation of the RANK level in the IT area vs. control (Fig. 1B, Tukey's test, p-value 0.0008), indicating an accumulation of osteoclast precursors. Moreover, a significant increase in the relative content of OPG (Fig. 1C, Tukey's test, p-value 0.012), osteocalcin (Fig. 1D, Tukey's test, p-value 0.021) and total RANKL (Fig. 2B, Tukey's test, p-value 0.009) proteins (by 1.48, 1.42, and 1.46 folds, respectively) was demonstrated as well, that could correlate, at least partially, with the high activity of osteoblasts and/or its precursors in the area of IT.
Fig. 2 Immunoblotting analysis of RANKL in bone tissue samples: representative immunoblots are shown (A) and quantified using β-actin as a loading control. The bar graphs of the total content of RANKL (B), its membrane-bound (C) and soluble (D) forms are shown as arithmetic means ± SEM (n = 6/group): 1 – bone tissue of healthy persons (control group); 2 – bone tissue, that was taken near the healthy tooth in patients with tooth impaction; 3 – bone tissue, collected near the impacted tooth. Experiments were performed in triplicate and *p<0.05 denotes significance compared with control, #p<0.05 denotes significance compared with bone tissue taken near the healthy tooth in patients with tooth impaction.
Physiological effects of OPG and RANKL on bone tissue metabolism are known to be largely determined by the ratio of their synthesis [13]. Based on the measurement of protein levels of RANKL/RANK/OPG signaling pathway components by western blot analysis, we obtained the ratios applied for assessing the status of local bone remodeling in the areas near the healthy and impacted teeth in patients with tooth impaction. It was established a significant decrease in the RANK/RANKL (2.6-fold) ratio and an increase in RANKL/OPG (2.5-fold) ratio in the area near the impacted tooth compared with the healthy tooth (Table 2). Declined RANK/RANKL ratio in the IT area may reflect the disturbances in the ligand-to-receptor binding and indicate the reduced effectiveness of signal transduction in the RANKL/RANK/OPG axis in addition to elevated RANKL/OPG ratio. Based on these observations we can assume that the local interrelation between bone resorption and bone formation in the IT area may lead to delayed tooth eruption/tooth impaction.
Table 2 Ratios of the RANKL/RANK/OPG signaling pathway components in the bone tissue of patients, in a.u. (arbitrary units)
Ratio, a.u.
|
Bone tissue, collected near the healthy tooth in patients with tooth impaction
|
Bone tissue, collected near the impacted tooth
|
RANK
|
0.97±0.06
|
1.73±0.11*
|
RANKL
|
0.32±0.02
|
1.46±0.12*
|
OPG
|
0.8±0,07
|
1.48±0.13*
|
RANK/RANKL
|
3.03
|
1.18*
|
RANKL/OPG
|
0.4
|
0.99*
|
* p<0.05 vs. bone tissue taken near the healthy tooth in patients with tooth impaction.
NF-κB-mediated signaling and apoptosis in the bone tissue
Binding RANKL to RANK leads to the further activation of NF-κB and translocation of this transcription factor to the nucleus to stimulate osteoclastogenesis [14]. To address whether excessive RANKL and RANK levels in bone tissue near the area of tooth impaction are engaged in the downstream transcriptional activation, we explored the involvement of NF-κB in the mechanisms of tooth eruption.
We demonstrated a slight decrease in the NF-κB (Fig. 3A, B, Tukey's test, p-value 0.043) and NFATc1 (Fig. 3A, C, Tukey's test, p-value 0.038) levels by 17 and 20% respectively in the bone tissue selected near the healthy tooth compared vs. control patients. Interestingly, it was demonstrated for the first time that despite the activation of the RANKL/RANK/OPG system in the impaction area, the content of both NF-κB and NFATc1 did not significantly change compared with the control group (Tukey's test, p-value >0.05), indicating a blocked or delayed process of osteoclastogenesis near the IT although an accumulation of osteoclast precursors in this area was observed.
Fig. 3 Immunoblotting analysis of NF-κB, NFATc1, and caspase-3 in bone tissue samples: representative immunoblots are shown (A, D) and normalized to β-actin. The bar graphs of NF-κB (B), NFATc1 (C), procaspase-3 (E) and its active subunit p17 (F) are demonstrated as arithmetic means ± SEM (n = 6/group): 1 – bone tissue of healthy persons (control group); 2 – bone tissue, that was taken near the healthy tooth in patients with tooth impaction; 3 – bone tissue, collected near the impacted tooth. Experiments were performed in triplicate and *p<0.05 denotes significance compared with control, #p<0.05 denotes significance compared with bone tissue taken near the healthy tooth in patients with tooth impaction.
Since caspase cascades play a key role in the activation of cell apoptosis [15] and may be triggered by NF-κB, we determined the levels of procaspase-3 (32 kDa) and its active subunit p17 (17 kDa). It was shown a decrease in the content of procaspase-3 (32 kDa) in the area of a healthy tooth of patients with tooth impaction by 28% (Tukey's test, p-value 0.031), while in the area of impacted tooth its content was increased by 1.32 folds compared with control (Fig. 3E, Tukey's test, p-value 0.028). Сonsidering the active caspase-3 enzyme is a heterodimer consisting of two p17 and two p11 caspase-3 subunits [16], it was also important to evaluate the content of the activated p17 form. We detected an increase of its protein level in the area of the healthy tooth by 2.26 folds compared vs. control (Tukey's test, p-value 0.0007), and an elevation in the area of the impacted tooth by 1.78 folds (Fig. 3F, Tukey's test, p-value 0.001) on the background of an accumulation of inactive procaspase-3 32 kDa. These observations indicate the cleavage of procaspase-3 and its activation in the area of the healthy tooth of patients with IT, that promote the tooth eruption process. We also suggest the impairment of caspase-3 activation and the accumulation of an inactive form in the area of tooth impaction may lead to tooth eruption failure.
Our data demonstrate that tooth impaction may be associated with blocked NF-κB signaling and NFATc1 expression and with the disturbances in the cascade activation of caspase-3.