PTH1R signalling regulates the mechanotransduction process of cementoblasts under cyclic tensile stress

PTH1R signalling regulates the mechanotransduction process of cementoblasts under cyclic tensile... Summary Objective To investigate the regulatory role of type I parathyroid hormone receptor (PTH1R) signalling in the mechanotransduction process of cementoblasts under cyclic tensile stress (CTS). Materials and methods Immortalized cementoblast cell line OCCM-30 were employed and subjected to cyclic tensile strain applied by a four-point bending system. The expression of PTHrP and PTH1R, as well as cementoblastic transcription factor Runx-2, Osterix, and extracellular matrix protein COL-1 and OPN were assessed by quantitative real-time polymerase chain reaction and western blot analysis. PTH1R expression was knocked down by siPTH1R transfection, and the alteration of cementoblastic biomarkers expression was examined to evaluate the function of PTH1R. Furthermore, to investigate possible downstream molecules, expression of signal molecule ERK1/2 with or without siPTH1R transfection, and the effect of ERK inhibitor PD98059 on the expression of cementoblastic biomarkers was also examined. Results Cyclic tensile strain elevated the expression of PTHrP and PTH1R, as well as cementoblastic biomarkers Runx-2, Osterix, COL-1, and OPN in a time-dependent manner, which was inhibited by siPTH1R transfection. The expression of phosphorylated ERK1/2 was upregulated time-dependently under cyclic stretch, which was also inhibited by siPTH1R transfection, and pretreatment of p-ERK1/2 inhibitor PD98059 undermined the increase of Runx-2, Osterix, COL-1, and OPN prominently. Conclusion The findings of the present study indicate that PTH1R signalling plays a regulatory role in the CTS induced cementoblastic differentiation in mature cementoblasts, and ERK1/2 is essentially involved as a downstream intracellular signal molecule in this mechanotransduction process. Introduction Root resorption induced by orthodontic treatment, is a common pathological phenomenon characterized by the destruction of cementum or even dentin layer of the root, which usually presented in the apical area as obtuse shortening of the root contour (1). The orthodontic factors influencing the prevalence of root resorption include tooth extraction, usage of multi-loop edgewise arch wire appliances and elastics, treatment duration, and tooth movement distance (2). All these factors induce root resorption depending on the magnitude, frequency, and duration of the orthodontic loading (3, 4). During orthodontic tooth movement, the mechanical stimuli perceived by periodontal ligament cells, including cementoblasts, were transduced into intracellular signalling, and elicits root resorption and repair via secretion of cementoclastic or cementoblastic molecules, which direct the cementoclast or cementoblast differentiation and recruitment (5, 6). The critical role of cementoblasts in orthodontically induced root resorption and repair is implicated by their function of cementum formation and property of mechanical responsiveness in vivo and in vitro (7). In most cases, mechanical strain, whether tensile or compressive, elicits cementoblastic differentiation in cementoblasts, whereas inhibitory effects on cementum formation is also reported under static compressive force (8–10). However, insufficient information is available on the mechanotransduction process of cementoblasts. Parathyroid hormone-related protein (PTHrP) is a local factor that mediates a variety of biological events such as skeletal development, in an autocrine/paracrine manner, through interaction with its receptor parathyroid hormone type I receptor (PTH1R) (11). The PTHrP–PTH1R system plays an essential role in the physiological process of tooth eruption and root formation. Dysfunction mutation of PTHrP would result in abnormal alveolar bone encasing the growing tooth germ in PTHrP KO mice (12), and deletion of PTH1R in dental mesenchymal progenitors is associated with eruption failure and truncated roots lacking periodontal ligaments due to accelerated cementoblastic differentiation (13). In clinical practice, orthodontic traction is usually applied to induce eruption of impacted teeth. Clinical reports have demonstrated that for patients with primary failure of eruption (PFE) associated with heterozygous mutation in intron 9 of the PTH1R gene, orthodontic traction usually leads to frustrating results even with assistance of surgical procedures (14). The above in vivo observations suggested the essential role of PTH1R in root development and mechanical responsiveness of periodontal ligament tissues. In vitro experiments identified PTH1R expression in cementoblasts, and the direct regulation role of PTHrP in osteoclastogenesis and extracellular matrix gene expression of cementoblasts towards mineralization inhibition is illustrated in previous studies (15, 16). Previous studies on chondrocytes revealed that mechanical stimulation upregulated PTHrP expression selectively in chondrocytes at the early maturational stage (17, 18). Despite the pivotal role of PTHrP and PTH1R in the dynamic process of root development in vivo and cementum formation in vitro, little is known about their functions in orthodontically induced root resorption and repair, and the possible downstream intracellular signalling pathways. Numerous studies have shown that mechanical forces initiated MAPK signal pathway in the periodontal tissues (19, 20). Among the MAPK signals, ERK1/2 was the most prominent kinase activated by various mechanosensors in regulating cell survival, differentiation and proliferation (21, 22). In particular, ERK1/2 signalling has been demonstrated to be involved in the cellular proliferation and differentiation during tooth root formation and regeneration (23, 24). However, the role of ERK signalling in the mechanotransduction process of cementoblasts during orthodontic treatment remains to be elucidated. The present study aims to investigate the cementoblastic activity of cementoblasts under cyclic tensile stress (CTS), and the regulatory role of PTH1R signalling in the mechanotransduction process. Furthermore, the function of the ERK signalling as the downstream mediator of PTH1R was also investigated. Materials and methods Cell culture Immortalized murine cementoblasts, OCCM-30 cells, were kindly donated by Professor Somerman (NIH, Bethesda, USA). OCCM-30 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin (Gibco, Carlsbad, California, USA) at 37°C in a humidified atmosphere of 5% CO2. Cyclic tensile stress Cyclic tensile stress was administrated using a four-point bending apparatus as reported previously (8, 25). OCCM-30 cells were seeded on the loading plates at a concentration of 4 × 105 cells/cm2. After the cells reached 80% confluence, the cells were serum deprived for 24 h with serum-free medium. Subsequently, the cells were exposed to CTS (0.5Hz, 2000 μstrain) for 1, 6, 12, and 24 h, respectively. In the control experiments, cells cultured on loading plates were kept in the same incubator without mechanical stress. Samples were collected at each time point, and each group was repeated in triplicate. The following are described in detail in the Supplementary text, table, and images: PTH1R siRNA transfection Real-time quantitative PCR (RT-PCR) Western blotting Statistical analysis All data are presented as the mean ± standard deviation (SD) of three independent experiments. Data were examined for normal distribution and homogeneity of variance using SPSS v.13.0 (Chicago, Illinois). We used one-way analysis of variance (ANOVA) to evaluate the statistical differences for the multiple comparisons and used independent t test to compare the expression levels between only two groups. Statistical comparisons were considered significant when P < 0.05. Results Cyclic tensile stress promotes cementoblastic activity in cementoblasts The expression of transcription factor Runx2 and Osterix, and cementogenic extracellular matrix protein osteopontin (OPN) and collegan type I (COL-1) were examined at the mRNA and protein level, respectively. Runx2 and Osterix mRNA expression was upregulated over time, with a robust rise at 6 h for Runx2 and at 12 h for Osterix, respectively. The highest expression level of Runx2 and Osterix at 24 h was approximately 5–6 times of the expression level in the unloaded group. OPN mRNA expression was significantly augmented at 6 h of tensile stress (4.54-fold) followed with a gradual down-regulation trend. COL-1 mRNA expression was elevated at 1 h, with a little setback at 6 h before a steady increase till 24 h (7.18-fold) (Figure 1A). Consistently, western blot assay demonstrated similar increase patterns of the cementoblastic/osteoblastic biomarkers at the protein level (Figure 1B). Figure 1. View largeDownload slide Effect of cyclic tensile stress on Runx2, Osterix, OPN, and COL-1 expression in cementoblasts. (A) Relative mRNA levels of Runx2, Osterix, OPN, and COL-1 examined by RT-PCR. (B) Western blotting and quantitative results of Runx2, Osterix, OPN, and COL-1 expression at the protein level. Data are represented as mean ± standard deviation (SD) (n = 3); &P < 0.05, *P < 0.01 versus control. Figure 1. View largeDownload slide Effect of cyclic tensile stress on Runx2, Osterix, OPN, and COL-1 expression in cementoblasts. (A) Relative mRNA levels of Runx2, Osterix, OPN, and COL-1 examined by RT-PCR. (B) Western blotting and quantitative results of Runx2, Osterix, OPN, and COL-1 expression at the protein level. Data are represented as mean ± standard deviation (SD) (n = 3); &P < 0.05, *P < 0.01 versus control. PTHrP and PTH1R expression increased during cyclic stretching-induced cementoblast differentiation Under cyclic tensile strain, PTHrP gene expression demonstrated a robust increase at 6 h and maintained at a higher level of expression 6–8 times of the control group until 24 h. PTH1R gene expression exhibited a steady increase from 1 to 24 h, and peaked at 24 h (3.03-fold) (Figure 2A). Protein expression patterns corroborated the results in gene expression of both PTHrP and PTH1R (Figure 2B). Figure 2. View largeDownload slide Effect of cyclic tensile stress on PTHrP and PTH1R expression in cementoblasts. (A) Relative mRNA levels of PTHrP and PTH1R by RT-PCR. (B) Western blotting and quantitative results of PTHrP and PTH1R at the protein level. Data are represented as mean ± SD (n = 3); *P < 0.01 versus control. Figure 2. View largeDownload slide Effect of cyclic tensile stress on PTHrP and PTH1R expression in cementoblasts. (A) Relative mRNA levels of PTHrP and PTH1R by RT-PCR. (B) Western blotting and quantitative results of PTHrP and PTH1R at the protein level. Data are represented as mean ± SD (n = 3); *P < 0.01 versus control. PTH1R silencing inhibits cyclic stretching-induced cementoblastic activity in cementoblasts To further testify the critical role of PTH1R signalling in the cyclic stretching-induced cementoblast differentiation, siRNA was constructed specifically for PTH1R to knock down PTH1R expression in OCCM-30 cells. The gene and protein expression of PTH1R were remarkably inhibited (69.1 ± 4.8%, 55 ± 4.5%) after PTH1R siRNA transfection and remained below the control group throughout the 24-h loading period (P < 0.01; Figure 3A). The qRT-PCR and western blot analysis revealed that, after PTH1R silencing, the cyclic tension induced upregulation of cementoblastogenic transcription factor Runx2, Osterix, and matrix protein OPN, COL-1 was significantly suppressed. (Figure 3). Figure 3. View largeDownload slide Effect of siPTH1R on Runx2, Osterix, COL-1, and OPN expression under cyclic tensile stress in cementoblasts. (A) RT-PCR analysis of PTH1R, Runx2, Osterix, COL-1, and OPN mRNA expression at 0, 1, 6, and 24 h of tension. (B) Western blot results of PTH1R, Runx2, Osterix, COL-1, and OPN proteins at 0, 1, 6, and 24 h of tension. Data are represented as mean ± SD (n = 3); #P < 0.05, &P < 0.01 versus siPTH1R control, $P < 0.05, *P < 0.01 versus corresponding scrambled. Figure 3. View largeDownload slide Effect of siPTH1R on Runx2, Osterix, COL-1, and OPN expression under cyclic tensile stress in cementoblasts. (A) RT-PCR analysis of PTH1R, Runx2, Osterix, COL-1, and OPN mRNA expression at 0, 1, 6, and 24 h of tension. (B) Western blot results of PTH1R, Runx2, Osterix, COL-1, and OPN proteins at 0, 1, 6, and 24 h of tension. Data are represented as mean ± SD (n = 3); #P < 0.05, &P < 0.01 versus siPTH1R control, $P < 0.05, *P < 0.01 versus corresponding scrambled. ERK mediates tensile stress induced cementoblast differentiation downstream of PTH1R signalling To further explore the possible downstream mechanotransductive signal involved in the PTH1R regulated cyclic tensile stress-induced cementoblast differentiation, we examined the expression of p-ERK and ERK under mechanical loading with or without siPTH1R. As expected, phosphorylation-ERK expression was increased in a time-dependent manner under mechanical stretch, with the peak level at 24 h (3.3-fold) (P < 0.05; Figure 4A). The siPTH1R transfection suppressed the mechanical induced increase of p-ERK (P < 0.05), which was later partly restored after 24 h of mechanical loading. Further, to testify whether ERK1/2 mediates tensile stress induced cementoblast differentiation downstream of PTH1R signalling, OCCM-30 cells were pretreated with p-ERK1/2 inhibitor PD98059 for 30 min before subjected to cyclic tensile stress. The activation of ERK under cyclic tensile stress was significantly inhibited by PD98059 administration, concomitant with attenuated upregulation of cementoblastic/osteoblastic biomarkers Runx2, Osterix, COL-1, and OPN (P < 0.01). The results indicated that ERK1/2 might be involved as a downstream pathway of the PTH1R signalling in mechanically induced cementoblast differentiation in mature cementoblasts (Figure 4B). Figure 4. View largeDownload slide Role of pERK1/2 as a downstream regulator of PTH1R in cyclic tensile stress-induced expression of Runx2, Osterix, COL-1, and OPN. (A) Western blots and quantitative results of p-ERK1/2 and ERK1/2 under cyclic tension with or without siPTH1R. (B) Western blot analysis of the expression of Runx2, Osterix, COL-1, and OPN proteins at 24 h with pretreatment of p-ERK1/2 inhibitor PD98059. Data are represented as mean ± SD (n = 3); *P < 0.05 versus ctrl or scrambled, #P < 0.01 versus stress group, &P < 0.01 versus no stress group. Figure 4. View largeDownload slide Role of pERK1/2 as a downstream regulator of PTH1R in cyclic tensile stress-induced expression of Runx2, Osterix, COL-1, and OPN. (A) Western blots and quantitative results of p-ERK1/2 and ERK1/2 under cyclic tension with or without siPTH1R. (B) Western blot analysis of the expression of Runx2, Osterix, COL-1, and OPN proteins at 24 h with pretreatment of p-ERK1/2 inhibitor PD98059. Data are represented as mean ± SD (n = 3); *P < 0.05 versus ctrl or scrambled, #P < 0.01 versus stress group, &P < 0.01 versus no stress group. Discussion Orthodontically induced root resorption (OIRR) is an unavoidable pathologic side effect concomitant with orthodontic tooth movement. Although cementum is commonly regarded to possess a higher anti-resorptive property and lower mineral-remodeling potential than alveolar bones under orthodontic stress, the incidence of root resorption is relatively high in clinical practice (26). Therefore, interpretation of the mechanotransduction process and biochemical response of cementoblasts under mechanical loading is conducive to elucidation of the underlying mechanisms of OIRR. Considering the greater resistance of cementum tissues to resorption than alveolar bones, the cementoblasts were speculated to be hypo-mechanosensitive in some previous studies, but the assumption remains controversial (27). The mechanical responsiveness of cementoblasts is confirmed by microscopic observations of new cementum deposition on the tensile side in orthodontic patients, and several in vitro loading experiments on cementoblasts (7–10). The mechanotransduction property of cementoblasts resembles the major characteristics of osteoblasts, but differed in the gene expression profile with a higher OPG/RANKL ratio under mechanical compressive stress (27, 28). The discrepancy between cementoblasts and osteoblasts in mechanical biological response may account for the greater resistance to resorption in cementum than alveolar bones under orthodontic compression. In the present study, the expression of cementoblastic transcription factor Runx2, Osterix and subsequently extracellular matrix protein COL-1, OPN was significantly upregulated in a time-dependent manner under cyclic stretching. The expression of cementoblastic biomarkers was quantified as relative level to the housekeeping gene GAPDH. Although GAPDH was used as reference for western blotting and RTqPCR as common practice, limitations are reported for normalization of gene expression in many cases dependent on cell density, differential expression across species, tissue types, cell lines (29, 30). However, previous studies proved that the mRNA and protein expression of GAPDH, served as reference gene, remained stable and at high levels under mechanical stimulation in OCCM-30 cells (9, 31, 32). Therefore, the present study employed GAPDH as reference gene. Furthermore, in order to avoid the interference of experimental conditions, cells were seeded on the plates at the same density (4 × 105 cells/cm2) and cultured at the same atmosphere with or without mechanical stimulation. Mechanical stimulation in vitro includes a variety of forms, such as static compression, fluid shear strain, or vibration. In the present study, we employed a four-point bending system to mimic the strained condition in periodontal ligament tissues in clinical orthodontic treatment (8, 9), which has been applied to investigate the mechanobiology of cells for many years (33). The force magnitude was set at 2000 μstrain, which is widely considered equivalent to physiological loading in vivo and clinical optimum orthodontic force by previous studies (8,9); and the force interval was set at 0.5Hz, in order to minimize the turbulent fluid flow over the cell surface during bending, according to previous studies (9, 25). This four-point bending system exerts strains on the cells’ substrate in uniform distribution and uniaxial direction, and the magnitude could be quantified through strain gauging. However, like all the other in vitro models, this four-point bending system could not replicate the complex strained conditions and interactions of cells in vivo. Previous studies have shown that the uniaxial tensile or compressive force, within the physiological intensity, promotes cementoblast differentiation (8, 9, 32). And this phenomenon was further testified by the present study. Such cyclic stretching induced upregulation of Runx2 and Osterix in cementoblasts was also detected in a previous study under 6 and 12% elongation in a force magnitude-dependent manner (34). Recent studies have been investigating the molecular mechanisms behind the mechanotransduction process of cementoblasts, signal molecules including inflammatory factor IL-1β, cytokine TNF-α, and miR-146b-5p and its target gene Smad4 have been reported to be involved in this process (8, 34–36). Osterix, as detected in the present study that remarkably increased under cyclic tension, is reported to regulate differentiation of precementoblasts via inhibition of DKK-1 and activation of Wnt/β-catenin signal pathway (37, 38). However, further investigation of more mechanotransductive molecules in cementoblasts would contribute to wider comprehension of the underlying mechanism. PTHrP was first identified in malignant hypercalcemia as a peptide molecule that mimicked parathyroid hormone. Further researches have revealed its widespread distribution in body, and its endocrine, paracrine, and autocrine modes of physiologic action as a central role in organogenesis, including tooth germ development and tooth eruption (11, 39). PTHrP mutation usually causes tooth impaction and eruption failure (12, 14). Deletion of PTH1R in osterix-expressing mesenchymal progenitors in dental follicle leads to eruption failure and truncated roots absent of periodontal ligament, which involves uncontrolled cementoblast differentiation (13). All these in vivo transgenic animal models verified the pivotal role of PTHrP-PTH1R system in tooth eruption and root development, including cementum generation. In the present study, PTHrP and PTH1R expression was upregulated prominently under mechanical stretching in a time-dependent manner, consistent with the expression pattern of cementoblastic/osteoblastic biomarkers Runx2, Osterix, and OPN, COL-1. Knocking down of PTH1R with siRNA transfection partly suppressed the mechanical induction of the cementoblastic/osteoblastic transcriptor Runx2, Osterix, and OPN, COL-1. These results confirmed the essential role of PTHrP-PTH1R system in the mechanotransduction process of cementoblasts under cyclic tensile stress. Previous studies have identified mechanical induced PTHrP expression predominantly in chondrocytes dependent on the maturation stage, which is approximately consistent with the experimental results of cementoblast loading in the present study (17, 18, 40). In vitro studies on cementoblasts also testified the negative regulatory effect of PTHrP on extracellular matrix gene expression and biomineralization, as well as the facilitative effect of PTHrP on cementoclastogenesis of cementoblasts (15, 16). These results seem to be contradictory to the present result that PTHrP increased concomitant with cementoblastic/osteoblastic biomarkers Runx2, Osterix, OPN, and COL-1. In fact, the biphasic effect of PTHrP on the differentiation and mineralization of osteoblastic cells has been confirmed by former researches concerning bone metabolism, and the ultimate effect of PTHrP is determined by the administration pattern (40, 41). Intermittent low-dose PTHrP enhances osteoblastic differentiation and mineralization, whereas continuous high-dose PTHrP inhibits this process (40, 41). This biphasic effect of PTHrP on osteoblastogenesis might partially explain the present result. Another in vitro study also suggested the role of PTHrP as a candidate mediator of the anabolic effect of mechanical loading on osteoblasts, which indirectly accorded with the present study (42). Previous studies have testified the anabolic effect of PTH on cementum formation of cementoblasts after mechanical stimulation (31), which is achieved through interaction between exogenous PTH and its receptor PTH1R on cell membrane. However, in the present study, PTH1R was demonstrated to mediate the mechanotransduction process independent of exogenous PTH or PTHrP administration. And the role of PTH1R signalling in this process probably involves two aspects, one is the autocrine PTHrP-PTH1R interaction, and the other is the mechanosensor function of PTH1R as an independent ion channel. In the present study, knocking down of PTH1R by siRNA transfection led to suppression of the mechanical induced upregulation of the cementoblastic/osteoblastic biomarkers Runx2, Osterix, and OPN, COL-1. The result verified the relationship between PTH1R expression and cementoblastic/osteoblastic biomarker upregulation under mechanical strain. As a receptor distributed extensively in bone tissues, PTH1R has been proven to amplify the osteogenic response to mechanical loading in vivo (43). Despite its role as a receptor to PTHrP, PTH1R has also been identified as a mechanosensor in osteocytes partially independent of PTHrP, potentiating calcium influx of mechanical activated cation channels (44, 45). Activation of PTH1R facilitates gap junction-mediated intercellular coupling, and couples to multiple intracellular signal transducers, including mitogen-activated protein kinases (MAPKs) (46). Extracellular signal-regulated kinase (ERK1/2) is an essential member of MAPKs, and modulates mechanotransduction in osteoblasts (21). The possible role of ERK as a mechanical transducer downstream of PTH1R activation in cementoblasts has been corroborated in the present study, as the mechanical enhanced expression of phosphorylated ERK was notably repressed by PTH1R silencing, and ERK inhibitor PD98059 blocked mechanical induction of cementoblast differentiation. Previous studies have reported the bidirectional effect of exogenous PTHrP on p-ERK1/2 expression in osteoblastic linage cells depending on the differentiation stage (47). In the present study, p-ERK expression increased concomitant with PTHrP, indicating an enhancement effect of PTHrP on p-ERK expression in mature cementoblasts. The aforementioned biphasic effect of PTHrP on p-ERK may be due to the distinct active conformations of PTH1R, which couple with diverse intracellular signalling pathways, including PKA and PKC mediated G-protein dependent pathway, β-arrestin mediated G-protein independent pathway, and calcium influx through PTH1R as an independent mechanosensor (44, 48, 49). However, the intracellular pathways responsible for the signal transduction between PTH1R activation and p-ERK upregulation remain to be further elucidated. Conclusion In summary, the findings of the present study suggested the essential regulatory role of PTH1R signalling in the cementoblastic/osteoblastic activity of mature cementoblasts under cyclic tensile stress, and ERK1/2 was indicated as a downstream signal during this process. These concepts are illustrated in the model depicted in Figure 5. The present study provides new insights into the molecular mechanisms behind the mechanotransduction process of cementoblasts, and contributes to better comprehension of root resorption and repair during orthodontic tooth movement. Figure 5. View largeDownload slide Proposed mechanism for the role of PTH1R in the cementoblastic differentiation regulated by cyclic tensile stress in cementoblasts. Figure 5. View largeDownload slide Proposed mechanism for the role of PTH1R in the cementoblastic differentiation regulated by cyclic tensile stress in cementoblasts. Supplementary material Supplementary material are available at European Journal of Orthodontics online. Funding This work was supported by the National Nature Science Foundation of China (grant number 81500880 [F.L], 81470777 [S.J.Z]), Beijing Natural Science Foundation (7172086) and Beijing Municipal Administration of Hospitals’ Ascent Plan DFL (20151401 [Y.X.B]). Conflict of interest None declared. References 1. 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( 2016) Binding selectivity of abaloparatide for PTH-Type-1-receptor conformations and effects on downstream signaling. Endocrinology , 157, 141– 149. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The European Journal of Orthodontics Oxford University Press

PTH1R signalling regulates the mechanotransduction process of cementoblasts under cyclic tensile stress

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Abstract

Summary Objective To investigate the regulatory role of type I parathyroid hormone receptor (PTH1R) signalling in the mechanotransduction process of cementoblasts under cyclic tensile stress (CTS). Materials and methods Immortalized cementoblast cell line OCCM-30 were employed and subjected to cyclic tensile strain applied by a four-point bending system. The expression of PTHrP and PTH1R, as well as cementoblastic transcription factor Runx-2, Osterix, and extracellular matrix protein COL-1 and OPN were assessed by quantitative real-time polymerase chain reaction and western blot analysis. PTH1R expression was knocked down by siPTH1R transfection, and the alteration of cementoblastic biomarkers expression was examined to evaluate the function of PTH1R. Furthermore, to investigate possible downstream molecules, expression of signal molecule ERK1/2 with or without siPTH1R transfection, and the effect of ERK inhibitor PD98059 on the expression of cementoblastic biomarkers was also examined. Results Cyclic tensile strain elevated the expression of PTHrP and PTH1R, as well as cementoblastic biomarkers Runx-2, Osterix, COL-1, and OPN in a time-dependent manner, which was inhibited by siPTH1R transfection. The expression of phosphorylated ERK1/2 was upregulated time-dependently under cyclic stretch, which was also inhibited by siPTH1R transfection, and pretreatment of p-ERK1/2 inhibitor PD98059 undermined the increase of Runx-2, Osterix, COL-1, and OPN prominently. Conclusion The findings of the present study indicate that PTH1R signalling plays a regulatory role in the CTS induced cementoblastic differentiation in mature cementoblasts, and ERK1/2 is essentially involved as a downstream intracellular signal molecule in this mechanotransduction process. Introduction Root resorption induced by orthodontic treatment, is a common pathological phenomenon characterized by the destruction of cementum or even dentin layer of the root, which usually presented in the apical area as obtuse shortening of the root contour (1). The orthodontic factors influencing the prevalence of root resorption include tooth extraction, usage of multi-loop edgewise arch wire appliances and elastics, treatment duration, and tooth movement distance (2). All these factors induce root resorption depending on the magnitude, frequency, and duration of the orthodontic loading (3, 4). During orthodontic tooth movement, the mechanical stimuli perceived by periodontal ligament cells, including cementoblasts, were transduced into intracellular signalling, and elicits root resorption and repair via secretion of cementoclastic or cementoblastic molecules, which direct the cementoclast or cementoblast differentiation and recruitment (5, 6). The critical role of cementoblasts in orthodontically induced root resorption and repair is implicated by their function of cementum formation and property of mechanical responsiveness in vivo and in vitro (7). In most cases, mechanical strain, whether tensile or compressive, elicits cementoblastic differentiation in cementoblasts, whereas inhibitory effects on cementum formation is also reported under static compressive force (8–10). However, insufficient information is available on the mechanotransduction process of cementoblasts. Parathyroid hormone-related protein (PTHrP) is a local factor that mediates a variety of biological events such as skeletal development, in an autocrine/paracrine manner, through interaction with its receptor parathyroid hormone type I receptor (PTH1R) (11). The PTHrP–PTH1R system plays an essential role in the physiological process of tooth eruption and root formation. Dysfunction mutation of PTHrP would result in abnormal alveolar bone encasing the growing tooth germ in PTHrP KO mice (12), and deletion of PTH1R in dental mesenchymal progenitors is associated with eruption failure and truncated roots lacking periodontal ligaments due to accelerated cementoblastic differentiation (13). In clinical practice, orthodontic traction is usually applied to induce eruption of impacted teeth. Clinical reports have demonstrated that for patients with primary failure of eruption (PFE) associated with heterozygous mutation in intron 9 of the PTH1R gene, orthodontic traction usually leads to frustrating results even with assistance of surgical procedures (14). The above in vivo observations suggested the essential role of PTH1R in root development and mechanical responsiveness of periodontal ligament tissues. In vitro experiments identified PTH1R expression in cementoblasts, and the direct regulation role of PTHrP in osteoclastogenesis and extracellular matrix gene expression of cementoblasts towards mineralization inhibition is illustrated in previous studies (15, 16). Previous studies on chondrocytes revealed that mechanical stimulation upregulated PTHrP expression selectively in chondrocytes at the early maturational stage (17, 18). Despite the pivotal role of PTHrP and PTH1R in the dynamic process of root development in vivo and cementum formation in vitro, little is known about their functions in orthodontically induced root resorption and repair, and the possible downstream intracellular signalling pathways. Numerous studies have shown that mechanical forces initiated MAPK signal pathway in the periodontal tissues (19, 20). Among the MAPK signals, ERK1/2 was the most prominent kinase activated by various mechanosensors in regulating cell survival, differentiation and proliferation (21, 22). In particular, ERK1/2 signalling has been demonstrated to be involved in the cellular proliferation and differentiation during tooth root formation and regeneration (23, 24). However, the role of ERK signalling in the mechanotransduction process of cementoblasts during orthodontic treatment remains to be elucidated. The present study aims to investigate the cementoblastic activity of cementoblasts under cyclic tensile stress (CTS), and the regulatory role of PTH1R signalling in the mechanotransduction process. Furthermore, the function of the ERK signalling as the downstream mediator of PTH1R was also investigated. Materials and methods Cell culture Immortalized murine cementoblasts, OCCM-30 cells, were kindly donated by Professor Somerman (NIH, Bethesda, USA). OCCM-30 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin (Gibco, Carlsbad, California, USA) at 37°C in a humidified atmosphere of 5% CO2. Cyclic tensile stress Cyclic tensile stress was administrated using a four-point bending apparatus as reported previously (8, 25). OCCM-30 cells were seeded on the loading plates at a concentration of 4 × 105 cells/cm2. After the cells reached 80% confluence, the cells were serum deprived for 24 h with serum-free medium. Subsequently, the cells were exposed to CTS (0.5Hz, 2000 μstrain) for 1, 6, 12, and 24 h, respectively. In the control experiments, cells cultured on loading plates were kept in the same incubator without mechanical stress. Samples were collected at each time point, and each group was repeated in triplicate. The following are described in detail in the Supplementary text, table, and images: PTH1R siRNA transfection Real-time quantitative PCR (RT-PCR) Western blotting Statistical analysis All data are presented as the mean ± standard deviation (SD) of three independent experiments. Data were examined for normal distribution and homogeneity of variance using SPSS v.13.0 (Chicago, Illinois). We used one-way analysis of variance (ANOVA) to evaluate the statistical differences for the multiple comparisons and used independent t test to compare the expression levels between only two groups. Statistical comparisons were considered significant when P < 0.05. Results Cyclic tensile stress promotes cementoblastic activity in cementoblasts The expression of transcription factor Runx2 and Osterix, and cementogenic extracellular matrix protein osteopontin (OPN) and collegan type I (COL-1) were examined at the mRNA and protein level, respectively. Runx2 and Osterix mRNA expression was upregulated over time, with a robust rise at 6 h for Runx2 and at 12 h for Osterix, respectively. The highest expression level of Runx2 and Osterix at 24 h was approximately 5–6 times of the expression level in the unloaded group. OPN mRNA expression was significantly augmented at 6 h of tensile stress (4.54-fold) followed with a gradual down-regulation trend. COL-1 mRNA expression was elevated at 1 h, with a little setback at 6 h before a steady increase till 24 h (7.18-fold) (Figure 1A). Consistently, western blot assay demonstrated similar increase patterns of the cementoblastic/osteoblastic biomarkers at the protein level (Figure 1B). Figure 1. View largeDownload slide Effect of cyclic tensile stress on Runx2, Osterix, OPN, and COL-1 expression in cementoblasts. (A) Relative mRNA levels of Runx2, Osterix, OPN, and COL-1 examined by RT-PCR. (B) Western blotting and quantitative results of Runx2, Osterix, OPN, and COL-1 expression at the protein level. Data are represented as mean ± standard deviation (SD) (n = 3); &P < 0.05, *P < 0.01 versus control. Figure 1. View largeDownload slide Effect of cyclic tensile stress on Runx2, Osterix, OPN, and COL-1 expression in cementoblasts. (A) Relative mRNA levels of Runx2, Osterix, OPN, and COL-1 examined by RT-PCR. (B) Western blotting and quantitative results of Runx2, Osterix, OPN, and COL-1 expression at the protein level. Data are represented as mean ± standard deviation (SD) (n = 3); &P < 0.05, *P < 0.01 versus control. PTHrP and PTH1R expression increased during cyclic stretching-induced cementoblast differentiation Under cyclic tensile strain, PTHrP gene expression demonstrated a robust increase at 6 h and maintained at a higher level of expression 6–8 times of the control group until 24 h. PTH1R gene expression exhibited a steady increase from 1 to 24 h, and peaked at 24 h (3.03-fold) (Figure 2A). Protein expression patterns corroborated the results in gene expression of both PTHrP and PTH1R (Figure 2B). Figure 2. View largeDownload slide Effect of cyclic tensile stress on PTHrP and PTH1R expression in cementoblasts. (A) Relative mRNA levels of PTHrP and PTH1R by RT-PCR. (B) Western blotting and quantitative results of PTHrP and PTH1R at the protein level. Data are represented as mean ± SD (n = 3); *P < 0.01 versus control. Figure 2. View largeDownload slide Effect of cyclic tensile stress on PTHrP and PTH1R expression in cementoblasts. (A) Relative mRNA levels of PTHrP and PTH1R by RT-PCR. (B) Western blotting and quantitative results of PTHrP and PTH1R at the protein level. Data are represented as mean ± SD (n = 3); *P < 0.01 versus control. PTH1R silencing inhibits cyclic stretching-induced cementoblastic activity in cementoblasts To further testify the critical role of PTH1R signalling in the cyclic stretching-induced cementoblast differentiation, siRNA was constructed specifically for PTH1R to knock down PTH1R expression in OCCM-30 cells. The gene and protein expression of PTH1R were remarkably inhibited (69.1 ± 4.8%, 55 ± 4.5%) after PTH1R siRNA transfection and remained below the control group throughout the 24-h loading period (P < 0.01; Figure 3A). The qRT-PCR and western blot analysis revealed that, after PTH1R silencing, the cyclic tension induced upregulation of cementoblastogenic transcription factor Runx2, Osterix, and matrix protein OPN, COL-1 was significantly suppressed. (Figure 3). Figure 3. View largeDownload slide Effect of siPTH1R on Runx2, Osterix, COL-1, and OPN expression under cyclic tensile stress in cementoblasts. (A) RT-PCR analysis of PTH1R, Runx2, Osterix, COL-1, and OPN mRNA expression at 0, 1, 6, and 24 h of tension. (B) Western blot results of PTH1R, Runx2, Osterix, COL-1, and OPN proteins at 0, 1, 6, and 24 h of tension. Data are represented as mean ± SD (n = 3); #P < 0.05, &P < 0.01 versus siPTH1R control, $P < 0.05, *P < 0.01 versus corresponding scrambled. Figure 3. View largeDownload slide Effect of siPTH1R on Runx2, Osterix, COL-1, and OPN expression under cyclic tensile stress in cementoblasts. (A) RT-PCR analysis of PTH1R, Runx2, Osterix, COL-1, and OPN mRNA expression at 0, 1, 6, and 24 h of tension. (B) Western blot results of PTH1R, Runx2, Osterix, COL-1, and OPN proteins at 0, 1, 6, and 24 h of tension. Data are represented as mean ± SD (n = 3); #P < 0.05, &P < 0.01 versus siPTH1R control, $P < 0.05, *P < 0.01 versus corresponding scrambled. ERK mediates tensile stress induced cementoblast differentiation downstream of PTH1R signalling To further explore the possible downstream mechanotransductive signal involved in the PTH1R regulated cyclic tensile stress-induced cementoblast differentiation, we examined the expression of p-ERK and ERK under mechanical loading with or without siPTH1R. As expected, phosphorylation-ERK expression was increased in a time-dependent manner under mechanical stretch, with the peak level at 24 h (3.3-fold) (P < 0.05; Figure 4A). The siPTH1R transfection suppressed the mechanical induced increase of p-ERK (P < 0.05), which was later partly restored after 24 h of mechanical loading. Further, to testify whether ERK1/2 mediates tensile stress induced cementoblast differentiation downstream of PTH1R signalling, OCCM-30 cells were pretreated with p-ERK1/2 inhibitor PD98059 for 30 min before subjected to cyclic tensile stress. The activation of ERK under cyclic tensile stress was significantly inhibited by PD98059 administration, concomitant with attenuated upregulation of cementoblastic/osteoblastic biomarkers Runx2, Osterix, COL-1, and OPN (P < 0.01). The results indicated that ERK1/2 might be involved as a downstream pathway of the PTH1R signalling in mechanically induced cementoblast differentiation in mature cementoblasts (Figure 4B). Figure 4. View largeDownload slide Role of pERK1/2 as a downstream regulator of PTH1R in cyclic tensile stress-induced expression of Runx2, Osterix, COL-1, and OPN. (A) Western blots and quantitative results of p-ERK1/2 and ERK1/2 under cyclic tension with or without siPTH1R. (B) Western blot analysis of the expression of Runx2, Osterix, COL-1, and OPN proteins at 24 h with pretreatment of p-ERK1/2 inhibitor PD98059. Data are represented as mean ± SD (n = 3); *P < 0.05 versus ctrl or scrambled, #P < 0.01 versus stress group, &P < 0.01 versus no stress group. Figure 4. View largeDownload slide Role of pERK1/2 as a downstream regulator of PTH1R in cyclic tensile stress-induced expression of Runx2, Osterix, COL-1, and OPN. (A) Western blots and quantitative results of p-ERK1/2 and ERK1/2 under cyclic tension with or without siPTH1R. (B) Western blot analysis of the expression of Runx2, Osterix, COL-1, and OPN proteins at 24 h with pretreatment of p-ERK1/2 inhibitor PD98059. Data are represented as mean ± SD (n = 3); *P < 0.05 versus ctrl or scrambled, #P < 0.01 versus stress group, &P < 0.01 versus no stress group. Discussion Orthodontically induced root resorption (OIRR) is an unavoidable pathologic side effect concomitant with orthodontic tooth movement. Although cementum is commonly regarded to possess a higher anti-resorptive property and lower mineral-remodeling potential than alveolar bones under orthodontic stress, the incidence of root resorption is relatively high in clinical practice (26). Therefore, interpretation of the mechanotransduction process and biochemical response of cementoblasts under mechanical loading is conducive to elucidation of the underlying mechanisms of OIRR. Considering the greater resistance of cementum tissues to resorption than alveolar bones, the cementoblasts were speculated to be hypo-mechanosensitive in some previous studies, but the assumption remains controversial (27). The mechanical responsiveness of cementoblasts is confirmed by microscopic observations of new cementum deposition on the tensile side in orthodontic patients, and several in vitro loading experiments on cementoblasts (7–10). The mechanotransduction property of cementoblasts resembles the major characteristics of osteoblasts, but differed in the gene expression profile with a higher OPG/RANKL ratio under mechanical compressive stress (27, 28). The discrepancy between cementoblasts and osteoblasts in mechanical biological response may account for the greater resistance to resorption in cementum than alveolar bones under orthodontic compression. In the present study, the expression of cementoblastic transcription factor Runx2, Osterix and subsequently extracellular matrix protein COL-1, OPN was significantly upregulated in a time-dependent manner under cyclic stretching. The expression of cementoblastic biomarkers was quantified as relative level to the housekeeping gene GAPDH. Although GAPDH was used as reference for western blotting and RTqPCR as common practice, limitations are reported for normalization of gene expression in many cases dependent on cell density, differential expression across species, tissue types, cell lines (29, 30). However, previous studies proved that the mRNA and protein expression of GAPDH, served as reference gene, remained stable and at high levels under mechanical stimulation in OCCM-30 cells (9, 31, 32). Therefore, the present study employed GAPDH as reference gene. Furthermore, in order to avoid the interference of experimental conditions, cells were seeded on the plates at the same density (4 × 105 cells/cm2) and cultured at the same atmosphere with or without mechanical stimulation. Mechanical stimulation in vitro includes a variety of forms, such as static compression, fluid shear strain, or vibration. In the present study, we employed a four-point bending system to mimic the strained condition in periodontal ligament tissues in clinical orthodontic treatment (8, 9), which has been applied to investigate the mechanobiology of cells for many years (33). The force magnitude was set at 2000 μstrain, which is widely considered equivalent to physiological loading in vivo and clinical optimum orthodontic force by previous studies (8,9); and the force interval was set at 0.5Hz, in order to minimize the turbulent fluid flow over the cell surface during bending, according to previous studies (9, 25). This four-point bending system exerts strains on the cells’ substrate in uniform distribution and uniaxial direction, and the magnitude could be quantified through strain gauging. However, like all the other in vitro models, this four-point bending system could not replicate the complex strained conditions and interactions of cells in vivo. Previous studies have shown that the uniaxial tensile or compressive force, within the physiological intensity, promotes cementoblast differentiation (8, 9, 32). And this phenomenon was further testified by the present study. Such cyclic stretching induced upregulation of Runx2 and Osterix in cementoblasts was also detected in a previous study under 6 and 12% elongation in a force magnitude-dependent manner (34). Recent studies have been investigating the molecular mechanisms behind the mechanotransduction process of cementoblasts, signal molecules including inflammatory factor IL-1β, cytokine TNF-α, and miR-146b-5p and its target gene Smad4 have been reported to be involved in this process (8, 34–36). Osterix, as detected in the present study that remarkably increased under cyclic tension, is reported to regulate differentiation of precementoblasts via inhibition of DKK-1 and activation of Wnt/β-catenin signal pathway (37, 38). However, further investigation of more mechanotransductive molecules in cementoblasts would contribute to wider comprehension of the underlying mechanism. PTHrP was first identified in malignant hypercalcemia as a peptide molecule that mimicked parathyroid hormone. Further researches have revealed its widespread distribution in body, and its endocrine, paracrine, and autocrine modes of physiologic action as a central role in organogenesis, including tooth germ development and tooth eruption (11, 39). PTHrP mutation usually causes tooth impaction and eruption failure (12, 14). Deletion of PTH1R in osterix-expressing mesenchymal progenitors in dental follicle leads to eruption failure and truncated roots absent of periodontal ligament, which involves uncontrolled cementoblast differentiation (13). All these in vivo transgenic animal models verified the pivotal role of PTHrP-PTH1R system in tooth eruption and root development, including cementum generation. In the present study, PTHrP and PTH1R expression was upregulated prominently under mechanical stretching in a time-dependent manner, consistent with the expression pattern of cementoblastic/osteoblastic biomarkers Runx2, Osterix, and OPN, COL-1. Knocking down of PTH1R with siRNA transfection partly suppressed the mechanical induction of the cementoblastic/osteoblastic transcriptor Runx2, Osterix, and OPN, COL-1. These results confirmed the essential role of PTHrP-PTH1R system in the mechanotransduction process of cementoblasts under cyclic tensile stress. Previous studies have identified mechanical induced PTHrP expression predominantly in chondrocytes dependent on the maturation stage, which is approximately consistent with the experimental results of cementoblast loading in the present study (17, 18, 40). In vitro studies on cementoblasts also testified the negative regulatory effect of PTHrP on extracellular matrix gene expression and biomineralization, as well as the facilitative effect of PTHrP on cementoclastogenesis of cementoblasts (15, 16). These results seem to be contradictory to the present result that PTHrP increased concomitant with cementoblastic/osteoblastic biomarkers Runx2, Osterix, OPN, and COL-1. In fact, the biphasic effect of PTHrP on the differentiation and mineralization of osteoblastic cells has been confirmed by former researches concerning bone metabolism, and the ultimate effect of PTHrP is determined by the administration pattern (40, 41). Intermittent low-dose PTHrP enhances osteoblastic differentiation and mineralization, whereas continuous high-dose PTHrP inhibits this process (40, 41). This biphasic effect of PTHrP on osteoblastogenesis might partially explain the present result. Another in vitro study also suggested the role of PTHrP as a candidate mediator of the anabolic effect of mechanical loading on osteoblasts, which indirectly accorded with the present study (42). Previous studies have testified the anabolic effect of PTH on cementum formation of cementoblasts after mechanical stimulation (31), which is achieved through interaction between exogenous PTH and its receptor PTH1R on cell membrane. However, in the present study, PTH1R was demonstrated to mediate the mechanotransduction process independent of exogenous PTH or PTHrP administration. And the role of PTH1R signalling in this process probably involves two aspects, one is the autocrine PTHrP-PTH1R interaction, and the other is the mechanosensor function of PTH1R as an independent ion channel. In the present study, knocking down of PTH1R by siRNA transfection led to suppression of the mechanical induced upregulation of the cementoblastic/osteoblastic biomarkers Runx2, Osterix, and OPN, COL-1. The result verified the relationship between PTH1R expression and cementoblastic/osteoblastic biomarker upregulation under mechanical strain. As a receptor distributed extensively in bone tissues, PTH1R has been proven to amplify the osteogenic response to mechanical loading in vivo (43). Despite its role as a receptor to PTHrP, PTH1R has also been identified as a mechanosensor in osteocytes partially independent of PTHrP, potentiating calcium influx of mechanical activated cation channels (44, 45). Activation of PTH1R facilitates gap junction-mediated intercellular coupling, and couples to multiple intracellular signal transducers, including mitogen-activated protein kinases (MAPKs) (46). Extracellular signal-regulated kinase (ERK1/2) is an essential member of MAPKs, and modulates mechanotransduction in osteoblasts (21). The possible role of ERK as a mechanical transducer downstream of PTH1R activation in cementoblasts has been corroborated in the present study, as the mechanical enhanced expression of phosphorylated ERK was notably repressed by PTH1R silencing, and ERK inhibitor PD98059 blocked mechanical induction of cementoblast differentiation. Previous studies have reported the bidirectional effect of exogenous PTHrP on p-ERK1/2 expression in osteoblastic linage cells depending on the differentiation stage (47). In the present study, p-ERK expression increased concomitant with PTHrP, indicating an enhancement effect of PTHrP on p-ERK expression in mature cementoblasts. The aforementioned biphasic effect of PTHrP on p-ERK may be due to the distinct active conformations of PTH1R, which couple with diverse intracellular signalling pathways, including PKA and PKC mediated G-protein dependent pathway, β-arrestin mediated G-protein independent pathway, and calcium influx through PTH1R as an independent mechanosensor (44, 48, 49). However, the intracellular pathways responsible for the signal transduction between PTH1R activation and p-ERK upregulation remain to be further elucidated. Conclusion In summary, the findings of the present study suggested the essential regulatory role of PTH1R signalling in the cementoblastic/osteoblastic activity of mature cementoblasts under cyclic tensile stress, and ERK1/2 was indicated as a downstream signal during this process. These concepts are illustrated in the model depicted in Figure 5. The present study provides new insights into the molecular mechanisms behind the mechanotransduction process of cementoblasts, and contributes to better comprehension of root resorption and repair during orthodontic tooth movement. Figure 5. View largeDownload slide Proposed mechanism for the role of PTH1R in the cementoblastic differentiation regulated by cyclic tensile stress in cementoblasts. Figure 5. View largeDownload slide Proposed mechanism for the role of PTH1R in the cementoblastic differentiation regulated by cyclic tensile stress in cementoblasts. Supplementary material Supplementary material are available at European Journal of Orthodontics online. Funding This work was supported by the National Nature Science Foundation of China (grant number 81500880 [F.L], 81470777 [S.J.Z]), Beijing Natural Science Foundation (7172086) and Beijing Municipal Administration of Hospitals’ Ascent Plan DFL (20151401 [Y.X.B]). Conflict of interest None declared. References 1. 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The European Journal of OrthodonticsOxford University Press

Published: Jan 31, 2018

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