Calcitonin Induces Osteogenic Differentiation Of Human Periodontal Stem Cells By Promoting Phosphorylation Of CREB

BackgroundThe latest clinical results investigated that alveolar bone loss and even tooth defect caused by periodontitis is the main cause of tooth loss in Chinese adults.At present,the means and ideas for the treatment of periodontitis are relatively limited,so it is dramatically urgent to explore new methods for the treatment of periodontitis.Periodontitis is a kind of chronic inflammation of periodontal supporting tissue.Dental plaque caused by microorganisms can affect the absorption and repair of alveolar bone and break its normal physiological dynamic balance,thus reducing the height of alveolar bone.Therefore,the purpose of periodontitis treatment is not only to control and eliminate the inflammation,but also to regenerate the damaged periodontal tissue and produce new attachments as much as possible.At present,the focus of research is to restore the regeneration and repair of periodontal tissue to the level of natural teeth in terms of morphology and function.This goal is likely to be achieved based on the current rapid development of stem cell and tissue engineering technology.Therefore,the biological research on the specific mechanism of alveolar bone repair and targeted targeted therapy will have a very urgent need and very important significance in promoting periodontal tissue regeneration and restoring the height of alveolar bone.Periodontal ligament stem cell((periodontal ligament stem cells,PDLSCs)is a kind of stem cell with multi-directional differentiation potential,which exists in the mesenchyme of periodontal ligament.It plays an important role in the repair of tissue injury after periodontal disease.Originally,PDLSCs was obtained by enzyme digestion.Seo et al.found that this kind of cells isolated from human permanent periodontal tissue have the potential to differentiate into a variety of periodontal tissues.In addition,some studies showed that PDLSCs could express osteogenic related proteins such as OCN and BSP under the condition of induction and culture in vitro.at the same time,when PDLSCs bound to scaffold was implanted into animal model,periodontal tissue regeneration could be observed,indicating that PDLSCs has the potential to differentiate into osteoblasts.The above results in vitro and in vivo suggest that PDLSCs plays an important role in the repair of alveolar bone and is the cytological basis of alveolar bone regeneration.Exploring the mechanism of inducing osteogenic differentiation of PDLSCs is of inestimable value for periodontal tissue repair and regeneration.The current research results show that the osteogenic differentiation of PDLSCs is regulated by a variety of cytokines in the microenvironment of periodontal tissue.And one of them attracted our special attention.In recent years,calcitonin(calcitonin,CT),a kind of cytokine,has been paid more and more attention to in bone tissue repair and bone reconstruction.Calcitonin was first extracted and found in parathyroid gland by Copper et al in 1962.It is a mature polypeptide encoded from human chromosome 11 and translated into 32 amino acids.It has disulfide bonds at position 1 and 7 and terminal carboxyl amidation.It has a variety of physiological functions and is more and more widely used.It belongs to the same family as calcitonin-related peptide(CGRP)and adrenomedullin(ADM).Members of this family can activate intracellular signal pathways by binding to the same kind of receptors,thus affecting the biological properties of cells,and are widely involved in the regulation of physiological activities such as bone metabolism and calcium homeostasis.Tian et al.found that CGRP can promote the phosphorylation of transcription factor CREB through c AMP pathway,and then regulate osteogenic differentiation.Recent studies have shown that CT and its family members induce osteogenic differentiation of stem cells after fracture healing by promoting CREB phosphorylation.Wei has shown that CT plays an important role in the regeneration and repair of periodontitis and can promote the osteogenic differentiation of periodontal ligament fibroblasts((periodontal ligament fibroblasts,PDLFs).But so far,there is little research on the mechanism of osteogenic differentiation between CT and PDLSCs,but there may be great research prospects for CT-induced PDLSCs in alveolar bone repair and regeneration and periodontitis treatment.The purpose of this study is to explore the role and molecular mechanism of CT in inducing osteogenic differentiation of PDLSCs by promoting CREB phosphorylation.Objective1.To identify the role of calcitonin and CREB expression in the osteogenic differentiation of PDLSC.2.To clarify the relationship and mechanism between calcitonin and CREB expression and phosphorylation in the osteogenic differentiation of PDLSC.3.To clarify the mechanism of CREB promoting osteogenic differentiation of PDLSC by regulating the expression of OPN.Methods1.Human periodontal ligament cells were subcultured and separated by magnetic beads.Human periodontal ligament stem cells were identified by immunofluorescence staining and flow cytometry analysis.2.Construct recombinant adenovirus.Western blot detect osteopontin expression in PDLSCs infected with Ad.CT and calcium deposition was detected by alizarin red.To explore the role of CT expression in osteogenic differentiation of PDLSC.3.Construct recombinant adenovirus.Western blot detect osteopontin expression in PDLSCs infected with Ad.CREB,and manipulate alizarin red to detect calcium deposition in cells.To explore the role of CREB expression in osteogenic differentiation of PDLSC..4.Construct recombinant adenovirus.Western blot detect the expression of OPN in PDLSCs co-infected with Ad.CREB/OPNsi RNA,and detect the calcium deposition of cells alizarin red.To explore whether CT regulates PDLSCs calcification through CREB pathway.5.Construct recombinant adenovirus.Western blot detect CREB and p CREB expression of different time table in PDLSCs infected with Ad.CT,and manipulate alizarin red to detect calcium deposition in cells.To explore the relationship between the regulation of CREB expression and phosphorylation by CT and its temporal trend in osteogenic differentiation of PDLSC.6.Construct recombinant adenovirus.Western blot detect osteopontin expression in PDLSCs infected with Ad.CT,the binding of CREB to OPN promoter was detected by chromosome immunoprecipitation(Ch IP),the DNA sequence of OPN promoter region bound by CREB was verified by sequencing,and the transcriptional regulation of CREB on OPN was detected by double luciferase report experiment.To explore the molecular mechanism of CREB regulating OPN expression during osteogenic differentiation of PDLSC.Results1.The cells were subcultured and the antibody positive cells were sorted successfully.The result of flow measurement is that the proportion of STRO1+ is 16.7%and the proportion of CD146 + is 21.0%.Immunofluorescence staining showed that wavy filament protein staining was positive and keratin staining was negative.2.After adenovirus mediated transgene overexpressed CT in PDLSCs,alizarin red was used to detect the calcium deposition at different time points.The results showed that the alizarin red staining of the over expressed CT group was significantly deeper than that of the empty virus control group at 7 and 14 days.Western blot was used to detect the expression of osteogenic differentiation marker OPN.The results showed that the expression level of OPN in overexpression CT group was significantly higher than that in control group at 7 and 14 days.These results suggest that the osteogenic differentiation trend of PDLSCs after CT treatment is 7 days,and it is obvious at 14 days.3.After overexpression of CREB in PDLSCs by adenovirus mediated transgene,the calcium deposition of PDLSCs at different time was detected by alizarin red,and the expression level of CREB / p CREB / OPN was detected by Western blot.The results of Western blot showed that PDLSCs were infected Ad.CREB The expression of CREB was maintained at a high level at 1,3,7 and 14 days after infection,and was significantly higher than that of the empty virus control group.At the same time,there was no significant difference in the expression level of CREB at each time point.The expression level of p CREB was also significantly higher than that of the control group,but we noticed that the peak of p CREB expression appeared on the third day.Alizarin red staining showed that calcification was significant in CREB overexpression group at 7and 14 days compared with empty virus control group.Western blot results of OPN also showed that the protein expression level of OPN in CREB overexpression group was significantly higher than that in control group at 7 and 14 days.4.In order to explore whether CT regulates the calcification of PDLSCs through CREB,CREB inhibitor was added at the same time.Western blot results showed that CREB inhibitor had no significant effect on the expression of CREB,but significantly inhibited the formation of p CREB.The results showed that the alizarin red staining of CT / CREB inhibition group was significantly lighter than that of CT intervention group at 7 and 14 days.Western blot was used to detect the expression of osteogenic differentiation marker OPN.The results showed that the expression level of OPN in overexpression CT group was significantly higher than that in CT / CREB inhibition group at 7 and 14 days.These results suggest that the promoting effect of CT on osteogenic differentiation of PDLSCs can be blocked by CREB inhibitor.5.After overexpression of CT in PDLSCs,Western blot was used to detect the expression and phosphorylation of CREB at different time points.The results showed that the expression level of CREB in the experimental group was significantly higher than that in the control group 3 days after overexpression of CT,and the high expression level continued to 14 days;however,for p CREB,the expression level of p CREB in the experimental group was significantly higher than that in the control group 1 and 3 days after overexpression of CT,but there was no significant difference between the expression level of p CREB and the control group 7 and 14 days after overexpression of CT.The expression level of p CREB reached the peak on day 3 and then decreased to a stable high level.6.Chromatin immunoprecipitation showed that CREB could bind to-1403 CRE and-1870 and-76AP-1 in AP-1 promoter region of human OPN gene.Construction and identification of luciferase reporter plasmid containing human OPN gene AP-1 promoter sequence showed that CREB could promote the function of OPN gene AP-1 promoter,and promote the function of OPN gene AP-1 promoter through-1870 and-76AP-1.Conclusion1.The protein expression of OPN in PDLSCs after overexpression of CT increased significantly at 7 and 14 days,indicating that CT plays a role in the osteogenic differentiation of human PDLSCs.2.After overexpression of CREB in PDLSCs,the protein expression level of OPN increased significantly at 7 and 14 days,suggesting that CREB plays a role in the osteogenic differentiation of human PDLSCs.3.CT can promote the osteogenic differentiation of PDLSCs,but it can be blocked by CREB inhibitor.4.After overexpression of CT in PDLSCs,the expression level of CREB increased significantly at 3 days,and the high expression level continued to 14 days,but the expression level of p CREB reached the peak at 3 days,and then decreased to a stable high level.It is suggested that CT can promote the expression and phosphorylation of CREB,and the expression and phosphorylation of CREB change at different time points.5.CREB binds to-1403 CRE and-1870 and-76AP-1 in human OPN gene AP-1promoter region,and promotes the function of OPN gene AP-1 promoter through-1870and-76AP-1.