| Insufficient estrogen is believed to be one of the major causes of postmenopausal osteoporosis. Osteoporosis is considered as one of the risk factors for periodontal disease and tooth loss. A number of studies have suggested that estrogen may have an important role in chronic inflammatory periodontal diseases. It has been observed that gingival inflammation and hyperplasia frequently occur during puberty, pregnancy, and menstruation; gingival inflammation is also frequently observed in women taking oral contraceptives. However, the relationship between the levels of estrogen and the incidence and progression of periodontal diseases is yet to be clarified, partly because the precise effects of estrogen on periodontal tissues are not yet known.The periodontal ligament (PDL) is the connective tissue located between the alveolar bone and the root surface of the tooth. Cells of this ligament exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. They play an important role in maintaining the integrity of the periodontal tissue.Estrogen has been reported to increase alkaline phosphatase activity, the production of osteocalcin, and the formation of mineralized nodules in cultured PDL cells. Estrogen modulates the activity of target cells by binding specific intracellular estrogen receptors (ER). Two estrogen receptor subtypes, designated ERαand ERβ, have been identified. As estrogen plays an important role in maintaining normal bone turnover, the genes for ERαand ERβhave been considered as potential candidate genes that might influence bone mass and osteoporotic risk. In previous studies, PDL cells were shown to express the mRNA for ER. However, the effect of ER in estrogen-induced osteoblastic differentiation function of PDL cells are still poorly understood.In terms of this context we designed and carried out the experiment below in order to investigated the effect of ERβin estrogen-induced osteoblastic differentiation function of PDL cells, to help to find the signal transduction pathways that mediate cellular responses to estrogen and any possibilities of new countermeasures to avoid the alveolar bone resorption.1,The expression of ERs in hPDL cellsBy the culture of human periodontal ligament cells, we investigated ERs expression by RT-PCR and supported by western blot analysis.2,Construction of ERβsiRNA expression vector and its expression in hPDL cellsHairpin siRNA templates were designed based on ERβgene sequence and mRNA structure and were cloned into eukaryotic expression plasmid, then confirmation them by DNA sequencing. Using lipidosome method, hPDL cells were transfected with the siRNA recombinant vector. Stable clones cells were obtained after G418 screening. Inhibition effect of ERβmRNA and protein expression were detected by RT-PCR and western blot respectively. 3,Effect of ERβon the osteoblastic differentiation function of hPDL cellsStable transfected and nontransfected cells were cultured with a saturating concentration of 17β-estradiol (10-7 mol/L). ALP activity was analysed and the amount of OCN was assessed.4,Effect of ERβon OPG and RANKL expression in hPDL cellsStable transfected and nontransfected cells treated with or without 17β-estradiol (10-7 mol/L) for 48h. The protein and corresponding mRNA level of OPG and RANKL expression in the cells were quantitatively determined by western blot and RT-PCR.Results:1,After successful culture of hPDL cells, both ERαand ERβexpression were found in the cells by RT-PCR and western blot and the expression of ERβwere stronger than ERα.2,Eukaryotic expression plasmid expressing siRNA targeting ERβgene was constructed successfully. The results of the RT-PCR and western blot analysis showed that ERβmRNA and protein expression were inhibited and stabilized transfected cells were constructed successfully.3,Estradiol significantly enhanced the ALP activity and the production of OCN in hPDL cells. However, the ALP activity and the production of OCN in hPDL-siERβcells were not significantly changed after estradiol treatment.4,Estradiol caused an increase in OPG expression and decreased RANKL expression in hPDL cells. However, estradiol had no effect on the expression of OPG and RANKL in hPDL-siERβcells.Conclusion:1,Both ERαand ERβexpression were expressed in hPDL cells, providing the bases that the ERs may be involved in the pathology mechanism of alveolar bone loss in postmenopausal women.2,The ERβsiRNA plasmid was constructed successfully and ERβexpression of hPDL cells was inhibited by RNAi.3,ERβmay play important roles in estrogen-induced effects on osteoblastic differentiation function of hPDL cells and estrogen influences the bone formation capacity of hPDL cells mainly via ERβ.4,Estrogen may play an important role in exerting antiresorptive effects on alveolar bone, at least in part, by increasing the expression level of OPG versus that of RANKL via ERβin hPDL cells.
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