| Background and ObjectivesPeriodontitis is one of the two common diseases of oral cavity and the major cause of tooth loss. Periodontitis has high prevalence rate worldwide and exhibits even higher prevalence than caries in China. Periodontitis is a bacterial infectious inflammatory disease and characterized by the destruction of periodontal tissue. It can cause the pocket formation and alveolar bone resorption and finally lead to teeth loss. The ultimate goal of the periodontal treatment is to reconstruct the periodontium destroyed by periodontal disease and recover the function of periodontal tissue, which is periodontal regeneration. Alveolar bone regeneration plays an important role in periodontal regeneration. Bone reconstruction mainly depends on the mineralization of the bone matrix protein secreted by osteoblast and the proliferation and differentiation of stems cells play an important role in bone regeneration. Stem cells have the potency of self-renewal and differentiation, and the proliferation and differentiation of stem cells is affected by many mechanisms and microenvironment factors. Currently, growth factors and DNA sequence changing was used to regulate the osteogenic activity of stem cells. However, the price of growth factors is high and DNA sequence changing may cause irreversible damage. Epigenetic regulation aims to change the methylation and acetylation of histone or DNA, and then convert chromatin into a transcriptive active or inactive state. The epigenetic modification will not cause the change of chromatin DNA. Now very little research has been done about the epigenetic regulation on bone regeneration. Therefore, the current research aimed to detect the effect and mechanism of epigenetic regulation on the osteoblastic differentiation of BMSCs, and further study the effect of the epigenetic regulation on bone regeneration in vivo. This study will help us understand the role of epigenetic mechanism in osteoblastic differentiation of stem cells and provide a new idea for clinically developing new methods of bone regeneration.PHD finger protein8(PHF8) is a histone demethylase. It can act on multiple histones and regulate the mono-and di-methylation of histones, and then regulates gene expression on transcriptional level. PHF8bound through its PHD domain to H3K4me3nucleosomes and demethylate H3K9, H3K27and H4K20at the transcription start site (TSS) regions of active promoters and then regulate gene transcription. PHF8has been proved to be involved in various biological processes. It has been confirmed that PHF8regulated many cell cycle genes and affected cell migration and invasion in cancer. PHF8was also proved to control the expression of genes associated with cell adhesion and cytoskeleton organization such as RhoA, Racl and GSK3β. A lack of PHF8not only led to a cell cycle delay but also resulted in a disorganized actin cytoskeleton and impaired cell adhesion. Others confirmed that PHF8governed Retinoic Acid response in Acute Promyelocytic Leukemia. PHF8was overexpressed in prostate cancer and affected cell proliferation, migration. Besides, PHF8was also found to regulate rRNA synthesis via its histone H3K9me1/2demethylase activity. More importantly, PHF8was recently found to play a critical role in craniofacial and bone development. Using zebrafish model, researchers found that PHF8was mostly expressed in the head and jaw regions. Injection of a zPHF8morpholino caused abnormalities in craniofacial organs and wild-type, and zPHF8showed significant rescue of the craniofacial defects induced by the zPHF8morpholino. These important findings identified a critical role of PHF8in craniofacial development.Satb2(Special AT-rich sequence-binding protein2) is a DNA-binding protein that regulates chromatin organization and gene expression. As PHF8, Satb2is also expressed in branchial arches and osteoblast-lineage cells. Satb2-/-mice exhibited defects in osteoblast differentiation and function, resulting in delayed bone formation and mineralization. Satb2-/-embryos showed multiple craniofacial defects including a significant truncation of the mandible and a cleft palate. Previous studies clearly demonstrated that Satb2played pivotal roles in bone regeneration, suggesting that Satb2can be used as an ideal osteogenic transcription factor to overcome the hurdles in craniofacial regeneration. Unfortunately, the profiles of the epigenetic regulation of the Satb2expression during BMSCs osteogenic differentiation particularly in oral and craniofacial development are still barely unknown.These insightful studies described above indicate the similarities in the function between PHF8and Satb2during osteogenic differentiation. In our current study, we detected the epigenetic regulation of PHF8on satb2and the role of PHF8in osteoblast differention and calvarial bone regeneration, and determined whether PHF8regulate osteogenic differentiation via its demethylase activity on Satb2.Materials and Methods:1. The cellular localization and the expression pattern of PHF8in different tissues and the change of PHF8during osteogenic differentiation.Different tissues were collected from8-week old C57BL/6J mice and immunohistochemistry (IHC) staining was used to detect the expression pattern of PHF8in different tissues. MC3T3-E1cells were recovered and passaged. When the cells exhibited good status, we used immunocytofluorescent to detect the cellular localization of PHF8. Primary BMSCs were collected from4-week old C57BL/6J mice and passaged. The second passage of BMSCs were treated with osteogenic medium for1,3,7,10,14and21d, and then total RNA was extracted and real time PCR was used to detect the mRNA expression level of bone marker genes, PHF8and Satb2.2. The effects and the mechanism of PHF8on the osteogenic differentiation of mice BMSCs and MC3T3-E1cellsBMSCs and MC3T3-E1cells were infected with PHF8, PHF8shRNA and the corresponding empty vector, and then RNA and protein was extracted. RT-PCR and western blot was used to determine the gene and protein expression of bone markers. MC3T3-E1cells were treated with osteogenic medium for7d and10d, and chromatin immunoprecipitation (ChIP) method was used to detect if PHF8can bind to the TSS region of Satb2; BMSCs and MC3T3-E1cells were infected with PHF8, PHF8shRNA and the corresponding empty vector; ChIP was used to detect the change of the binding of histone H3K9mel on Satb2chromatin.3. The effects of silk scaffold combined with PHF8modified BMSCs on the bone repair of mice calvarial bone defectsBMSCs were infected with PHF8, PHF8shRNA and the corresponding empty vector, and then the cells were then combined with silk scaffold. Calvarial bone defect model was made on the8-week old mice and the silk scaffolds combined with modified BMSCs were implanted in the defect region. Five weeks after surgery, mice were scraficed and Micro CT, H&E staining, IHC staining and Real Time PCR were used to determine the effect of PHF8modified BMSCs on bone repair.Results1. The cellular localization and the expression pattern of PHF8in different tissues, and the change of PHF8during osteogenic differentiation.There were little PHF8expression in kidney, liver and muscle. In heart, only several cells were PHF8positive. In long bone and calvarial bone there were much more PHF8positive cells than that in other tissues. Importantly, PHF8positive cells were mainly distributed in the growth plate of long bone and the cranial suture. IHC staining found that most PHF8was localized in nucleus in MC3T3-E1cells. The mRNA expression of PHF8and Satb2increased parallelly during osteogenic differentiation.2. PHF8promoted osteoblastic differentiation of BMSCs and MC3T3-E1cells via its histone demethylase activity.PHF8overexpression could upregulate mRNA and protein expression of Satb2, OSX, Runx2, BSP and OC, while knockdown PHF8using shRNA downregulated the expression of all these bone markers. ChIP results confirmed that PHF8bound to the TSS region of Satb2and the binding of PHF8to the TSS region of Satb2increased when cells were treated with osteogenic medium. Another ChIP analysis found that knockdown of PHF8using shRNA up-regulated the binding of H3K9mel at the TSS region of Satb2.3. PHF8-modified BMSCs promoted the healing of mice calvarial bone defect.Five weeks after implanting, the mice were sacrificed. Micro CT results confirmed that PHF8-modified BMSCs promoted the healing of mice calvarial bone defects. We also performed H&E staining and analyzed the new bone formation in different groups and got the same results as Micro CT. Real Time PCR results indicated that the expression levels of bone markers were higher in PHF8-modified BMSCs group than those in other groups.Conclusions:1. PHF8is essentially expressed in osteoblastic cells of cranial suture and growth plate of long bone.2. PHF8plays a critical role in osteogenic differentiation.3. PHF8enhances osteogenic differentiation via modulating histone methylation states of master gene Satb2.4. PHF8promotes bone wound healing in mice, therefore, can be used as a therapeutic molecule for bone regeneration. |
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