| Postmenopausal osteoporosis (osteoporosis, OP) is a common and frequently-occurring disease for older women. Estrogen and progesterone deficiencies are well-known major contributors of postmenopausal osteoporosis development. Osteoporosis is regarded as one of the causes of periodontal disease and tooth loss. Clinical observations in postmenopausal women show that a higher frequency of loss of alveolar bone height as well as a low mandible bone density is accompanied by lower estrogen level. Similar to estrogen, the serum level of progesterone also reduces markedly after menopause. Recent experimental and clinical data have also demonstrated that progesterone plays a major role in protecting the skeleton. However, less information is available on the anabolic effects of progesterone on the remodeling of alveolar bone.Periodontal ligament (PDL) is non-mineralized connective tissue located between the alveolar bone and cementum. PDL plays a vital role in maintaining homeostasis of periodontal tissues by affecting coordinated balance between bone formation and bone resorption activity. Thus, PDL is the fundamental requirement for both tooth eruption and orthodontic tooth movement. The periodontal ligament cells (PDLCs) are considered as multipotential cells and a source of osteoblasts.As well as being the regulator of the integrity of the skeleton, progesterone has the potential effects on periodontal tissues. It is obvious that periodontal disease manifestations occur when an imbalance of progesterone takes place. However, it is still not clear whether progesterone receptor (PR) is expressed in hPDLCs and whether progesterone has effects on the proliferation and differentiation of hPDLCs. Therefore, the purpose of the present study is to detect the expression of PR in hPDLCs and explore the effects of progesterone on the proliferation and differentiation of hPDLCs in vitro. This research might lay the foundation for studying progesterone and the PR mechanism in the alveolar bone resorption and remodeling.We designed and carried out the experiment as follows:1. The expression of progesterone receptor in human periodontal ligament cellsThe hPDL tissue was obtained from the extracted healthy premolar for orthodontic reasons. We performed the primary culture of hPDLCs. The PR expression in hPDLCs was detected by immunocytochemistry and the reverse transcriptase polymerase chain reaction.2. The effect of progesterone on the proliferation of human periodontal ligament cellsThe hPDLCs were stimulated with progesterone (1, 10, 100 nM) for 1, 2, 3, 4, 5, 6, and 7 days. MTT assay was performed to assess the effects of progesterone on cell growth curve. Cell cycle analysis was performed to further test the effect of 100 nM progesterone on the proliferation rate of hPDLCs by means of flow cytometry on the third day. The untreated hPDLCs served as control group. We employed RU486, a specific progesterone receptor antagonist, as antagonist group.3. The effect of progesterone on the differentiation of human periodontal ligament cellsTo quantify the effects of progesterone on osteogenic differentiation of hPDLCs, alizarin red and ALP staining were performed in basic culture medium and osteogenic medium. In this experiment we also examined the effect of the progesterone on ALP activity and the expression of BSP, OCN, and OPN mRNA in hPDLCs, respectively.4. The effects of combined estrogen and progesterone on proliferation and differentiation of human periodontal ligament cells.Cells were cultured in basic culture medium supplemented with progesterone, 17β-estrodiol or progesterone plus 17β-estrodiol. The effects of hormones on the proliferation were determined by MTT assay, and its effects on the differentiation were determined by alizarin red staining and ALP activity assay.5. The effect of progesterone on the expression of c-fos,c-jun and Runx2 in human periodontal ligament cells.Cells were untreated or treated with 100 nM progesterone in the absence or presence of 100 nM RU486.The expression of c-fos, c-jun mRNA were determined by RT-PCR. The effect of the progesterone on the expression of Runx2 mRNA in hPDLCs was determined by Realtime RT-PCR.We found that:1. In this study we proved the presence of PR mRNA in hPDLCs by using the RT-PCR and PR protein expression in hPDLCs by immunocytochemistry. The positive signal was strongly expressed in the nuclei but weaker in cytoplasm of hPDLCs.2. Progesterone can promote the proliferation of hPDLCs. When RU486 was added, this action was blocked. The percentage of the cells in S + G2M phases was higher in progesterone-treated group than that in control group and progesterone + RU486 group.3. In normal medium, the obvious mineralized nodules were detected in all the groups. The results showed that there were a few mineralized nodules in control group and progesterone + RU486 group. The amount of mineralized matrix increased in the progesterone-treated group. In osteogenic medium, increasing alizarin red–positive area was detected in osteogenic group, but the alizarin red–positive area was obviously less than that in osteogenic + progesterone group. The alizarin red–positive area was obviously decreased in osteogenic + progesterone + RU486 group. We got the similar result in ALP staining. The data have indicated that the progesterone can promote the ALP activity and the expression of BSP, OCN, and OPN mRNA in time- and dose-dependent manner in hPDLCs.4. The proliferation of hPDLCs in control group was still lower than that in progesterone-treated group, estrodiol-treated group and estrogen- progesterone group. The difference among the groups treated with progesterone, estrodiol and estrodiol-progesterone is not significant. The alizarin red-positive area increased in the progesterone-estrogen group compared with progesterone-treated group and estrogen group, and the alizarin red-positive area was obviously increased in osteogenic + estrogen + progesterone group than that in osteogenic + estrogen and osteogenic + progesterone group. The results of ALP activity assay showed that the ALP activity was higher in estrogen-progesterone group compared with other three groups.5. The results showed that the expression of c-fos, c-jun, and Runx2 mRNA was markedly enhanced in 100 nM Progesterone-treated group compared with control group, but decreased in progesterone + RU486 group.In conclusion:1. Our results demonstrate that the PR is expressed in hPDLCs at both mRNA and protein level.2. Progesterone can stimulate the proliferation of the hPDLCs, and RU486 can block the effect of progesterone.3. Progesterone can stimulate the differentiation of the hPDLCs, and RU486 can block the effect of progesterone. This also strongly supports the view that progesterone effects are mediated by the PR.4. Estrogen and progesterone also can promote the proliferation and differentiation of hPDLCs. The effect of using estrogen and progesterone together on cell proliferation is not significantly different, compared with using estrogen and progesterone alone. Moreover, combined estrogen and progesterone can further promote the differentiation of hPDLCs compared with progesterone group and estrogen group.5. Progesterone also can upregulate the expression of c-fos,c-jun, and Runx2 mRNA. Our current study suggested that progesterone may exert its bone-sparing functions by increasing the expression level of c-fos,c-jun, and Runx2 mRNA via PR in hPDLCs.
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