| Background and objectiveDuring the orthodontic treatment, the teeth move towards expected direction because of the reconstruction of peridontal tissue. Periodontal ligament plays an important role in the transmission of orthodontical force from tooth to periodontal tissue. Periodontal ligament is dense connective tissue locating between tooth root and alveolar bone, which is consisted by cells and matrix. Periodontal ligament fibroblasts (PDLFs) is the predominant cell in consisting periodontal ligament, and is able to synthetise various kinds of cytokine to mediate its own cellular behavior such as proliferation, differentiation and apoptosis through signal transduction system, which contribute to the remolding and regeneration of periodontal system. The investigation of how does the orthodontic force transduce inside the PDLFs, as well as how does the orthodontic force affect the PDLFs cellular behavior is helpful in understanding the mechanism of periodontal remolding promoted by orthodontic force and in searching the most suitable mechanical environment that could sustain the periodontal stability.Osteoblasts play an important role in promoting the bone matrix secretion and mineralization. Core binding factor a 1 (Cbfal), also called runt related transcription factor 2 (Runx2), is accepted as one of the most important osteogenic transcription factors. The protein could bind to the cis-acting element that exist on a serious of osteogenic genes, thus promoting the transcription of the target genes. Even as a specific osteogenic transcription factor, the expression of Runx2 is highly confined in the mineral tissue and osteoblast cell line, but more and more studies have proved that the expression level of Runx2 is not linear correlated to the function and differentiation of osteoblasts, because the activation status of Runx2 also affect its roles potentially. Overall, Runx2 is greatly accepted as a hub in connecting extracellular factors that could induce osteodifferentiation and intracellular signalings that control osteoblast function and differentiation.Extracellular signal regulated kinase 1/2 (ERK1/2) is the most typical and the earliest to be found among the Mitogen-activated protein kinase (MAPK) family members. Activated by upstream kinases, ERK1/2 could then transmit the signal further toward nuclear and affect the expression or activation of downstream factors. Runx2 has been proved to be one of those downstream targets of ERK1/2 in osteoblasts, but it is still unknown whether the ERK1/2-Runx2 pathway can be activated in mastication stimulated PDLFs, and if it does involve in the osteodifferentiation of PDLFs after mastication stimulation, the molecular mechanism of which is further needed to be explored.Our present study first prove the ability of Runx2 to promote the osteodifferentiation of PDLFs through transfecting the primary cultured PDLFs with lentivirus vector containing type Ⅱ Runx2 gene. Then we simulate the orthodontic force by constructing the in vitro mechanical tension application model and demonstrate the expressions of Runx2 and other related osteogenes, as well as ERK1/2 pathway could all be activated in PDLFs by cyclic stretch application. Finally, we apply the stretch on both normal PDLFs and Runx2 overexpressed PDLFs, and inhibit the ERK1/2 pathway using specific inhibitor at the same time, in order to intervene both of the factors in ERK1/2-Runx2 pathway. Our study interpreted the relationship among those factors:orthodontic force stimulation, Runx2, ERK1/2 and osteogene expression, clarified the mechanism of the PDLFs osteodifferentiation through ERKl/2-Runx2 pathway by cyclic tension loading. Our study proposed the possible molecular mechanism through which the orthodontic force stimulation could promote the PDLF osteodifferentiation and further the entire periodontal tisuue osteogenic remodeling, aiming to provide some new idea in clinical orthodontical treatment.Major methods and results1. Isolation and identification of PDLFsPrimary PDLFs were isolated through tissue combined with enzyme digestion methord. PDLFs growth curve were made by counting cell number. The endochylema was proved to be eosinophilic and the nuclear was proved to be basophilous according to HE staining. Keratin positive and vimentin negative results proved that the cultured cells derived from mesenchymal tissue. The result of immunofluorescence staining of fibroblast specific protein 1 (FSP1) was positive.2. Construction of the stably transfected PDLFs with type II Runx2 and effect of Runx2 in inducing the osteodifferentiation of PDLFs.We construct the LV5-Runx2 vector using type II Runx2 gene segment and lentivirus vector LV5. The virus stock solution was collected, and the concentration measured by limiting dilution assay was 6x105TU/ml. After the preliminary experiment was conducted for the optimal selecting concentration of Puromycin, as well as the optimal MOI value, we finally got the cloned PDLF overexpressing Runx2. Realtime PCR and Western blot were performed to confrim that mRNA and protein level of Runx2 were all elevated in stably transfected PDLFs. Realtime PCR results demonstrated that mRNA levels of osteogenes SP7, OCN and BSP were all promoted in Runx2 overexpressed PDLFs, COL-1 and ALP mRNA levels were unaffected, and ATF4 mRNA level reduced slightly in Runx2 overexpressed PDLFs. Alizarin red stain exprement found that 3 weeks culture of Runx2 overexpressed PDLFs led to the apperance of calcium nodule. Those results above proved that Runx2 overexpression alone could promote the osteodifferentiation of PDLFs.3. Construction of the cyclic stretch stimulated PDLFs modelWe applied the cyclic stretch of 10% amplitude and 0.5HZ frequency on PDLFs in vitro by using the multichannel cellular mechanical loading system, in order to simulate the biological orthodontical force loading onto the PDLFs. The loading time was 1h,3h,6h,12h,18h and 24h and unloaded PDLFs served as control group. Realtime PCR was conducted to detect the dynamic mRNA levels of Runx2, SP7, OCN, BSP and ATF4 in different loading time. Western blot and immunoprecipitation were conducted to detect the protein level of Runx2, as well as the activation of Runx2 and ERK1/2 pathway in different loading time. Those results above confirmed that current stretch loding system could effectively induce the osteogenes transcriptional expressions, and also activate the phosphorylation of Runx2 protein and ERK1/2 pathway during mechanical loading.4. ERK1/2-Runx2 pathway mediate the osteodifferentiation of PDLFs induced by cyclic stretchBoth the normal PDLFs and Runx2 overexpressed PDLFs were subject to cyclic stretch for 3h, and the mRNA level of ATF4, SP7, OCN and BSP were detected by realtime PCR. The results of realtime PCR showed that the mechanical stimualtion and Runx2 overexpression had a cooperative role in inducing the transcription of those genes above. Subsequently, inhibiting ERK1/2 pathway by U0126 on both normal PDLFs and Ruxn2(+)PDLFs while conducting mechanical stimulation significantly reduced the transcripts of those genes above, comparing to the mechanical stimulation without blocking ERK1/2 pathway. Further, by studing the mRNA level and protein level of Runx2 in the cells, we found that cyclic stretch could promote the transcriptional and translational expression of Runx2 in PDLFs; when ERK1/2 pathway was inhibited, the mRNA level of Runx2 could decrease, while Ruxn2 protein level didn’t reduce evidently. These results proved that the mechanical stimulation could induce the Runx2 transcriptional expression through ERK1/2 pathway, but ERK1/2 has little effect on Runx2 translational regulation. By studying the phosphorylation level of Runx2 (p-Ruxn2), we found that the expression ofp-Runx2 is highly related to the mRNA levels of those target genes of Runx2. Our conclusion is that the mechanism of ERK1/2-Runx2 in mediating the mechanical stretch stimulated PDLFs osteodifferentiation is promoting the phosphorylated protein level of Runx2, which could elevate the transactivation function of Runx2 in order to increasing the target genes transcription. Further analysis of both Runx2 and ERK1/2 protein revealed that ERK1/2 exist in cytoplasm in resting cells, while Runx2 stays in nuclear; mechanical stimulation activate the ERK1/2 and promote them to translocate into unclear where they could form protein complex with Runx2, which could provide the condition for Runx2 to be phosphorylated; specific inhibition of ERK1/2 by U0126 prevented the stretch-induced activation of ERK1/2 as well as its nuclear translocation, thus abate its effect on Runx2 phosphorylation.Conclusion1. Runx2 could promote the osteodifferentiation of PDLFs by inducing the transcription of its target genes such as SP7, OCN and BSP2. Orthodontic force simulated by cyclic stretch could elevate the mRNA expressions of SP7, OCN and BSP, as well as both the mRNA and protein level of Runx2. Besides, the ERKl/2 and Runx2 could be activated during the cyclic stretch stimulation3. The ERK1/2-Runx2 pathway participates in mechanical stretch-stimulated PDLFs osteodifferentiation through increased level of p-Runx2 protein, thus promote transcriptional expressions of its target osteogene; total Runx2 protein level is not correlated with the transcription level of its target osteogene. Stretch activated and promote the nuclear translocation of ERK1/2, which complex with Runx2, which presumably create condition for Runx2 phosphorylation. Inhibition of ERK1/2 by U0126 prohibit the effect of stretch on ERK1/2 and thus the phosphorylation of Runx2, which affect the osteogenes expressions in stretch loaded PDLFs.Innovation and significanceThis present study confirmed that the ERK1/2-Runx2 pathway involves in the cyclic stretch induced PDLFs osteodifferentiation from cellular and molecular level. Our study proposed the idea that activated Ruxn2 rather than total Runx2 is the predominant factor in regulating the transcriptions of its target genes, while activated ERK1/2 could lead to increase in Ruxn2 mRNA level, as well as further complex and phosphorylate Runx2 protein after nuclear translocation. Those opinions above could provide some new fresh ideas regarding the periodontal tissue remolding during clinical orthodontic treatment. |
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