| Cementum is a thin and bone-like mineralized tissue covering root surface. Periodontal ligament fibers peneatrate into cementum in one side and anchor to the surrounding alveolar bone in the other side, thereby fix tooth to the alveoous socket. So the integrity of cementum is of vital importance for protecting pulp from external stimulation and tooth stability.Orthodontically induced inflammatory root resorption(OIIRR) which is mainly characterized by the destruction of cementum, is a quite common phenomena presented during orthodontic treatment; Some sever cases may lead to tooth hyper-mobility, or even tooth exfoliation. Interestingly, the destruction of cementum can also be observed in patients suffering from periodontitis. What is more interesting is that an elevated expression level of TNF-a was reported in both periodontitis and orthodontic treatment, and then we propose TNF-a might be a key factor contributing to cementum destruction under orthodontic and periodontitis condition.This study aimed to clarify the effects of TNF-a on cementoblast differentiation, mineralization, apoptosis, and proliferation, as well as the underlying signal transduction mechanisms. The study includes the following three parts:Part1Effects of TNF-a on the mineralization ability of cementoblastsObjective:To explore the effects of different concentration TNF-a on the mineralization ability of cementoblasts.Methods:An immortalized murine cementoblast cell line OCCM-30 (kindly provided by Dr. Martha J. Somerman) was used in this study. Cells were seeded to 24-well cell culture plate with a density of 7.5×104 cell/well. Mineral induction medium(containing 50?g/ml AA and 10mM B-GP) supplied with various concentration (0,1,10,20,30,40ng/ml)TNF-? was administrated. After 10 days' mineral induction, alizarin red staining was performed and cetylpyridinium chloride was used for the semi-quantification of mineral nodules.Results:In the control group, large amounts of mineral nodules were formed. Less mineral nodules were formed in all groups treated with TNF-a compared with the control group. Moreover, the amount of mineral module was much less as TNF-? concentration increased.Conclusion:TNF-? inhibited the mineralization ability of cementoblasts in a concentration dependent manner.Part 2Effects of TNF-? on cementoblast differentiation, apoptosis, and proliferationObjective:To investigate the effects of TNF-? on cementoblast differentiation, apoptosis, and proliferation.Methods:(1) OCCM-30 cells were treated with mineral induction medium(containing 50?g/ml AA and lOmM B-GP) supplied with various concentration (0,1,10,20,30,40ng/ml)TNF-a, after 7 days' treatment, cells were havested for ALP activity analysis. (2) OCCM-30 cells were treated with different concentration TNF-? (0,1,10,20,30,40ng/ml) for a certain period, or 20ng/ml TNF-? for different time lengths (0-24 hours), and then the expression of osterix, BSP, OCN, cleaved caspase-3 was examined by real-time PCR and western blot analysis. (3) OCCM-30 cells were treated with different concentration TNF-? (0,1,10,20,30, 40ng/ml) for 24 hours, and then the percentage of apoptosis cell was evaluated by flow cytometric staining.Results:(1) ALP activity of OCCM-30 was suppressed by TNF-? (0-40ng/ml) in a concentration dependent manner; Osterix was inhibited by TNF-? (0-40ng/ml) at both transcriptional and protein level in a concentration dependent manner; (2) Osterix was inhibited by TNF-? (20ng/ml) at both transcriptional and protein level in a time dependent manner; Both BSP mRNA and OCN mRNA were suppressed by TNF-? (0-40ng/ml) in a concentration dependent manner; (3) The expression of cleaved caspase-3 was enhanced in a concentration dependent manner after stimulation with TNF-? (0-40ng/ml) for 24 hours; The expression of cleaved caspase-3 was up-regulated in a time dependent manner after stimulation with TNF-? (20ng/ml) for 0-24 hours; (4) Flow cytometric staining results showed that the percentage of apoptosis cell increased as TNF-? concentration increased.Conclusion:TNF-? inhibited differentiation and induced apoptosis in cementoblasts in a concentration and time dependent manner.Part 3The signal transduction mechanisms involved in the regulation of differentiation and apoptosis under TNF-? stimulationObjective:To elucidate the role of p53?PP2Ac?Erk1/2?p38?JNK, PI3K-Akt and NF-?B pathways in TNF-?-inhibited differentiation and TNF-?-induced apoptosis in cementoblasts.Methods:(1) real-time PCR and western blot analysis were performed to evaluate the expression of p53?MDM2?p21?PP2Ac alpha?PP2Ac beta?p-Erk1/2?Erk1/2? p-p38?p38?p-JNK?JNK?p-Akt?Akt?p-p65?p65 after TNF-? treatment; (2) cells were pre-treated with p53 specific inhibitor PFT-?, p53 siRNA, or PP2Ac specific inhibitor OA before TNF-? administration,24 hours after TNF-a treatment, real-time PCR and western blot analysis were performed to investigate the expression osterix and cleaved caspase-3; (3) cells were pre-treated with p38 inhibitor SB203580, Erkl/2 inhibitor PD98059, JNK inhibitor SP600125, PI3K-Akt inhibitor LY294002, and NF-?B inhibitor PDTC before TNF-a administration,24 hours after TNF-? treatment, real-time PCR and western blot analysis were performed to investigate the expression osterix and cleaved caspase-3; (4) cells were pre-treated with p38 inhibitor, Erkl/2 inhibitor, JNK inhibitor, PI3K-Akt inhibitor, and NF-?B inhibitor before TNF-a administration,24 hours after TNF-? treatment, real-time PCR and western blot analysis were performed to investigate the expression p53 and MDM2.Results:(1) The expression of p53?MDM2?p21?PP2Ac alpha?PP2Ac beta was up-regulated by TNF-?; The expression of phosphorylated form of Erk1/2?p38?JNK? Akt?p65 were dramatically increased while their corresponding total amounts remained unchaged; (2) when cells were pre-treated with p53 specific inhibitor PFT-a or p53 siRNA, osterix was up-regulated while cleaved caspase-3 was down-regulated compared to only TNF-a treatment group; when cells were pre-treated with PP2Ac specific inhibitor OA, osterix remained unchanged while cleaved caspase-3 was irregularly fluctuated compared to only TNF-? treatment group? (3) when cells were pre-treated with specific inhibitors for p38, Erkl/2, JNK, PI3K-Akt, and NF-?B pathways respectively, osterix was even more inhibited while cleaved caspase-3 was even more increased compared to only TNF-? treatment group; (4) when cells were pre-treated with specific inhibitors for p38, Erkl/2, JNK, PI3K-Akt, and NF-?B pathways respectively, p53 and MDM2 were even more enhanced compared to only TNF-? treatment group; on the contrary, when cells were pre-treated with specific inhibitor for NF-?B pathway, p53 and MDM2 were decreased compared to only TNF-? treatment group.Conclusion:(1)p53?PP2Ac?Erk1/2?p38?JNK?PI3K-Akt and NF-?B pathways were all activated by TNF-a in cementoblasts; (2) The differentiation inhibition and apoptosis inducing effects of TNF-a on cementoblasts were conducted, at least partially conducted via p53; (3)unlike p53, Erkl/2, p38, JNK, PI3K-Akt, and NF-?B pathways acted in a negative-feedback way to the action of TNF-? on cementoblasts; (4)The negative-feedback effects of Erkl/2, p38, JNK, and PI3K-Ak pathways were facilitated through, or at least partially through their inhibitory actions on p53, but it is not so for the NF-?B pathway. |
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