| Background and ObjectivesPeriodontitis is one of the two common diseases of oral cavity and the major cause of tooth loss. It is a bacterial infectious inflammatory disease and characterized by peridontal pocket formation and alveolar bone resorption. The characteristics of periodontal disease is the inflammation and destroy of periodontal supportive tissue(alveolar bone, cementum and periodontal ligament. The ultimate goal of periodontal therapy is to reconstruct the periodontium destroyed by inflammation, restore the structure and function of periodontium and require in the insertion of collagen fibres into the newly formed bone and cementum, which is the real meaning of periodontal regeneration. However, the real meaning of periodontal regeneration should be the repeat of embryogenesis and morphogenesis of development process. Among it, the proliferation and differentiation of stem cells is the key factor to determine successful peridontal regeneration, and many internal mechanisms and microenviromental farors can influence the proliferation and differentiation of stem cells. Oral cavity is an environment contaminated by bacteria and periodontitis is a bacterial infectious disease。During the process of periodontal regeneration, stem cells are in the inflammatory microenvironment and inflammtory cytokines probably influence the regualtion of stem cell proliferation and differentiation. Most of the present research focus on osteoblastic cell lines and demonstrate that inflammatory cytokines could influence the expression of osteoblast-specific genes, the secretion of extracellular matrix and the apoptosis of osteoblasts. However, the previous studies did not elucidate the regulation effect of inflammatory signal pathway on the expression of bone-related genes during the process of stem cells differentiation towards osteoblasts. Therefore, in the pathological and repair process of periodontits, the study on the effect of chronic, long-term and high concentration of inflammatory microenvironment on the differentiation of adult stem cells towards osteobalsts will facilitate us to understand the biological characteristics of adult stem cells under inflammatory stimulation and result in finding a new therapy for treating periodontal diseases more effectively.Nuclear transcription factor NF-κB widely exists in eukaryotes, and it is a protein family composed by complex polypeptide subunits. As the pivot of signal transduction pathway, NF-κB is critical for the occurrence and development of immune and tumor, the regulation of cell apoptosis and embryogenesis, therefore it is a key nuclear transcription factor. NF-κB plays an important role in the cytokine-induced gene expression. The genes it regulates encode acute phase reactive proteins, cytokines, cell adhesion molecules, immune regulation molecules, virus tumor genes, growth factors, transcription and growth regulation factors. By regualtion the expression of multiple genes, NF-κB takes part in many biological process including immunoreaction, inflammatory response, cell apoptosis, tumorigenesis and osteogenic differentiation. The classical activated DNA bound NF-κB signal is a dimer (the dimer composed by p50and p65) while in the resting state, NF-κB locates in cytoplasm with the inactive form and it combines with an inhibitor IκB and forms a trimer as p50-p65-IκB. When cells are stimulated by inflammatory cytokines such as TNF and IL-1, IκB is released from the trimer, and translocation signal on p50subunit and binding site on p56subunit are exposed and make this dimer exhibits the activity of NF-κB and translocate from cytoplasm to nucleus, then combines withKB motif and finally regulates the transcription process. In the inflammatory periodontal microenvironmen, NF-κB regulates the transcription process and the expression of various upstream and downstream osteogenic genes through complicted mechanism.Recently, some researchers recommend the NF-κB pathway is involved in the osteoblast induction of BMP2and Runx2. Not only does BMP2induce the bone formation in vivo, but also promote Mesenchymal cells osteoblast differentiation.The impossible cause is that inflammatory factor inhibit the bone regeneration and osteoblastic differentiation induced by BMP2. At present most of research show that TNF-α promote the BMP2expression through NF-κB pathway,then BMP2activate the Smad pathway to increase the expression of downstream osteoblast target molecules.However,some reports show TNF-a inhibit the osteoblast diferentiation through the NF-κB pathway in osteoblasts.The comfirmation of the exactly mechanism will be benefit to increase the osteoblast induction in the recovery of the bone defect in inflammation circumstance.In the numerous osteoblast regulatory factors, Runx2is considered to be the master gene. In the mice which knocked off the Runx2gene,the mature osteoblasts and ossification disappeared absolutely.The immature osteoblasts observed to weakly express the ALP acivity did not express osteopontin (OPN) and osteocalcin (OC). Some evidences show TNF-a inhibit the pro-osteoblast differentiation by blocking the Runx2activity. The exactly mechanism is unknown, it recommend us that inflammatory factor maybe take action between Runx2and downstream effector molecules through the NF-κB pathway.Therefore, in the present study,we overexpressed p50/p65in ST2cell to activate the NF-κB pathway,overexpressed IκB in ST2cell to block the NF-κB pathway. In vitro,to observe whether overexpression of NF-κB pathway simulate the role of inflammatory cytokines to induce osteoblast differentiation, to observe whether blocking the NF-κB pathway may delete the effects of the inflammatory cytokines on different osteoblast pathway. Add inflammatory stimulation and/or osteoblast induction to the cell co-transfected different Dual-Luciferase report genes,then detect the fluorescence value,to investigate whether TNF-α regulated BMP-Smad, Runx2-BSP, Runx2-OSE pathway through the NF-κB pathway.Materials and Methods:1. The effects of TNF-α on the osteoblast gene expression of osteoblast-induced ST2cell,and the regulation of TNF-a to the process that BMP2,Runx2promote downstream Osteogenesiswith the Osteogenesis induced medium, we treated cell with TNF-α and/or BMP2; TNF-αand/or Runx2, then digest the cell,collect protein and total RNA,detect the expression of different osteoblast gene by RT-PCR. Investigate the effects of TNF-α on the osteoblast gene expression of osteoblast-orient induced ST2cell,to survey whether TNF-α regulate the process which BMP2,Runx2promote downstream Osteogenesis.2. The effect of TNF-α and its downstream NF-κB pathway on BMP2-Smad signal pathwayp65, p50,IκB,and its control vetor stablely transfect the ST2cell. Cells are treated with osteoblast induction medium, TNF-α and/or BMP2. Total RNA of cell was extracted. Detected the expression of the target osteoblast gene. SBE-luc and TK were transient co-transfect into the target cells. TNF-α and/or BMP2stimulate the cell.Detecting the fluorescence value,to assess whether the transcription regulation of BMP2to downsream gene was verify by TNF-α and/or blocking the NF-κB pathway.3. TNF-α was capable of inhibiting Runx2-BSP and Runx2-OSE signal pathway through the NF-κB pathwaypCMV5-Runx2, BSP-luc(or OSE-luc) and TK were transient co-transfect into the target cells,which include the ST2cell stablely transfected p65/p50/IκB/control vetor plasmid, respectively With/without TNF-a stimulation,detect the fluorescence value of different cell groups.Results1. The effect of TNF-α on the gene expression levels of BMP2and Runx2in ST2cells during ostegenic differentiationThe mineralization associated genes in ST2cells, a osteoprogenitor cell line, are expressed in a spatial and temporal manner. BMP2and Runx2are in the upstream with high expression in the early stage of osteogenic differentiation, while OC and BSP are in the downstream. During the osteogenic induction of ST2cell, TNF-α was able to increase the expression level of BMP2while inhibiting the levels of Runx2, OC,BSP.TNF-α can inhibit the function in different level, which BMP2and Runx2 promote the downstream osteoblast gene expression.2.The promotion of the expression levels of BMP2by TNF-α was achieved through the NF-κB pathway.but TNF-a can not influence the BMP-Smad signal pathway.In ST2cells that the NF-κB signalling was blocked, with osteogenic induction, the promotion of TNF-α on BMP2expression was eliminated. And this blockage did not affect the regulatory roles of BMP2in the expression of downstream genes, as well as the BMP-Smad signal pathway.In NF-κB overexpressed ST2cells, the expression levels of BMP2was significantly increased during osteogenic differentiatin. When exogenous BMP2was applied, the expression of downstream genes was enhanced, such as OC and BSP, but not so much as the control group does. Overexpressed NF-κB do not affect BMP-Smad pathway。3. Through NF-κB pathway, TNF-amodulate the Runx2-BSP signal pathway,do not activate OSE promoterThe cells which overexpressed p50/p65plasmid are able to downregulate the expression of Runx2in the osteogentic process of ST2. After NF-κB pathway are blocked by force expression of IκBα,it inhibit the effect which TNF-a inhibit the expression of Runx2.Overexpression of p50/p65,TNF-α stimulation,both of them can inhibit the activity of Runx2,which induced the transcription of9.0kb BSP promoter. Overexpression of IκBα eliminate the inhibition. Neither NF-κB pathway nor TNF-α stimulation effect the activation of Runx2to OSE promoter.Conclusions:1.TNF-α upregulate the expression of BMP2mRNA, but the inhibition of TNF-α toward the downstream osteogenesis of BMP2eliminate the upregulation.2. Through the NF-κB pathway, TNF-α increase the expression of BMP2and inhibit the osteoblast differentiation which BMP2induced. However, the inhibition isn't dependent on BMPs-Smad pathway.3. Through the NF-κB pathway, TNF-a decrease the expression of Runx2and inhibit the osteoblast differentiation which Runx2induced. Through NF-κB pathway,TNF-α inhibit the transcription of BSP promoter,but TNF-α fail to affect the transcription of OSE promoter,which Runx2activate.
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