High Levels of Glucose Induced the Caspase-3/PARP Signaling Pathway, Leading to Apoptosis in Human Periodontal Ligament Fibroblasts

Periodontitis is one of the main complications of diabetes mellitus, and much research has been conducted on their relationship. However, the mechanism by which high glucose levels induce damage of periodontal ligament fibroblasts is still unclear. In this study, we investigated the effects of high glucose levels on apoptosis in human periodontal ligament fibroblasts and the possible mechanisms involved. Human periodontal ligament fibroblasts were cultured in DMEM with normal glucose (5.5 mM) and high glucose (35 mM) levels for 6, 12, or 24 h. Apoptosis was studied by flow cytometry, caspase assays, fluorescent real-time PCR, and Western-blot analysis. The different durations of high glucose incubation induced a time-dependent increase of apoptosis and caspase-3 activity in cultured human periodontal ligament fibroblasts. In addition, concentrations of caspase-3 and its substrate PARP in cultured human periodontal ligament fibroblasts increased in a time-dependent manner. Furthermore, a caspase-3 inhibitor could prevent the high glucose–induced apoptosis in human periodontal ligament fibroblasts. These data indicate that high glucose induces a time- and caspase-3-dependent increase in apoptosis in cultured human periodontal ligament fibroblasts. These results elucidate the mechanism for the regulation of human periodontal ligament fibroblast apoptosis caused by high glucose.     

Desmosomal proteins in cultured and intact human periodontal ligament fibroblasts

This study examined the kinds of desmosomal proteins in the human periodontal ligament fibroblasts (PDLFs). The PDLFs obtained from young and older patients were cultured and the amounts of desmosomal proteins were measured by ELISA with antibodies to desmoplakins, desmogleins, and desmocollins. Cultured cells and tissue sections of the human periodontal ligament were immunostained with the same antibodies. Expression of desmosomal proteins in the PDLFs was clearly demonstrated both by ELISA and the immunohistochemical studies, suggesting the existence of desmosome-like junctions in the PDLFs. The junctions are considered to protect gap junctions in the PDLFs against cell transformation caused by cell contraction, which may relate to tooth eruption and repair of periodontal tissue, and/or strong occlusal forces. Statistically significant differences (P < 0.0001) in the expression of desmoplakins and desmogleins between younger and older patients were observed in this study.     

Growth factor modulation of mitogenic responses and proteoglycan synthesis by human periodontal fibroblasts

In order to understand the relationship between specific growth factors and matrix synthesis by periodontal cells, we have investigated the effects of platelet-derived growth factor BB (PDGF-BB), insulin-like growth factor-I (IGF-1), and growth hormone on DNA and proteoglycan synthesis by cultured human gingival and periodontal ligament fibroblasts in vitro. PDGF-BB and IGF-1, but not growth hormone, were mitogenic for both periodontal ligament fibroblasts and gingival fibroblasts, although the periodontal ligament cells responded more strongly. The mitogenic response was accompanied by alterations in expression of matrix proteoglycan mRNA. For both the gingival and periodontal ligament cells, there was a decrease in mRNA for decorin and an increase in mRNA for versican following exposure to IGF-1 and PDGF-BB. Although no change was seen in response to PDGF, biglycan mRNA level was increased by IGF-1 in periodontal ligament fibroblasts. With the gingival fibroblats, biglycan mRNA levels were unaffected by IGF-1, PDGF-BB, or growth hormone. These findings suggest variable responses of fibroblasts to growth factors depending upon anatomical site within the periodontium. Moreover, there appears to be a correlation between cell proliferation and the types of proteoglycan synthesised with decorin expression being suppressed, and versican being increased during fibroblast proliferation. J. Cell. Physiol. 174:353–361, 1998. © 1998 Wiley-Liss, Inc.     

Double expressions of connexin 43 and 32 in human periodontal ligament fibroblasts

The expressions of connexin 43 and 32 in cultured and intact human periodontal ligament fibroblasts (PDLFs) were examined using immunohistochemical methods, and western blot analysis was conducted with anti-connexin 43 and 32 in cultured PDLFs. The PDLFs both in cultured cells and tissue sections reacted with anti-connexin 43 and 32, and western blot analysis showed bands of approximately 43 kD and 27 kD reacted with anti-connexin 43 and 32 respectively, suggesting the existence of gap junctions in human PDLFs. In cultured PDLFs there were no reaction products of connexin 43 when the cells were not in contact with adjacent cells, but reaction products were increasingly observed with increases in cell-cell contacts. Different from connexin 43, the reaction products of connexin 32 were found in the cytoplasm, regardless of whether the cells were or were not in contact with adjacent cells. Further, the reaction activity of connexin 32 varied among PDLFs; some were strong, some moderate, and some weak. The expressions of connexin 43 and 32 in human PDLFs are suggested to be related to the regulation of two different functions of the PDLFs.     

<Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>-derived lipopolysaccharide-mediated activation of MAPK signaling regulates inflammatory response and differentiation in human periodontal ligament fibroblasts

Porphyromonas gingivalis (P.g.), which is a potential pathogen for periodontal diseases, contains lipopolysaccharide (LPS), and this endotoxin stimulates a variety of cellular responses. At present, P.g.-derived LPS-induced cellular responses in human periodontal ligament fibroblasts (PDLFs) are not well characterized. Here, we demonstrate that P.g-derived LPS regulates inflammatory responses, apoptosis and differentiation in PDLFs. Interleukin-6 (IL-6) and -8 (IL-8) were effectively upregulated by treatment of P.g.-derived LPS, and we confirmed apoptosis markers including elevated cytochrome c levels, active caspase-3 and morphological change in the presence of P.g.-derived LPS. Moreover, when PDLFs were cultured with differentiation media, P.g.-derived LPS reduced the expression of differentiation marker genes, as well as reducing alkaline phosphatase (ALP) activity and mineralization. P.g.-derived LPS-mediated these cellular responses were effectively abolished by treatment of mitogen-activated protein kinase (MAPK) inhibitors. Taken together, our results suggest that P.g.-derived LPS regulates several cellular responses via activation of MAPK signaling pathways in PDLFs.     

Function of Chemokine (CXC Motif) Ligand 12 in Periodontal Ligament Fibroblasts

The periodontal ligament (PDL) is one of the connective tissues located between the tooth and bone. It is characterized by rapid turnover. Periodontal ligament fibroblasts (PDLFs) play major roles in the rapid turnover of the PDL. Microarray analysis of human PDLFs (HPDLFs) and human dermal fibroblasts (HDFs) demonstrated markedly high expression of chemokine (CXC motif) ligand 12 (CXCL12) in the HPDLFs. CXCL12 plays an important role in the migration of mesenchymal stem cells (MSCs). The function of CXCL12 in the periodontal ligament was investigated in HPDLFs. Expression of CXCL12 in HPDLFs and HDFs was examined by RT-PCR, qRT-PCR and ELISA. Chemotactic ability of CXCL12 was evaluated in both PDLFs and HDFs by migration assay of MSCs. CXCL12 was also immunohistochemically examined in the PDL in vivo. Expression of CXCL12 in the HPDLFs was much higher than that in HDFs in vitro. Migration assay demonstrated that the number of migrated MSCs by HPDLFs was significantly higher than that by HDFs. In addition, the migrated MSCs also expressed CXCL12 and several genes that are familiar to fibroblasts. CXCL12 was immunohistochemically localized in the fibroblasts in the PDL of rat molars. The results suggest that PDLFs synthesize and secrete CXCL12 protein and that CXCL12 induces migration of MSCs in the PDL in order to maintain rapid turnover of the PDL.     

SFRP2 and Slug Contribute to Cellular Resistance to Apoptosis in Hypertrophic Scars

Hypertrophic scars (HS) are skin disorders which occur after wounding and thermal injury. Our previous studies have suggested that secreted frizzled-related protein 2 (SFRP2) is involved in HS formation and that the suppression of SFRP2 promotes apoptosis of hypertrophic scar fibroblasts (HSFBs). However, the mechanisms have not been clarified. Previous studies revealed that Slug expression inhibits cell apoptosis, in vitro and in vivo, and SFRP2 regulates the expression of Slug in cervical cancer cells. In the present study, we quantified differential expression levels of expression of SFRP2 and Slug in HS and normal skin tissues by immunohistochemistry, both of which have important anti-apoptosis roles. Furthermore, a short hairpin RNA approach was adopted to investigate the potential function of SFRP2 and Slug in HSFB apoptosis. Cell apoptosis was detected using fluorescence-activated cell sorting and Caspase-3 activity was assayed by spectrophotometry. This study demonstrates that SFRP2 expression, as well as Slug, is dramatically up-regulated in HS relative to normal skin tissues, and the Slug expression is positively correlated with SFRP2. Slug expression was down-regulated in SFRP2-deficient cells, and the down-regulation of Slug expression increased sensitivity to apoptosis which was induced through a caspase-3-dependent pathway. The infected cells with reduced levels of Slug were tested for the expression of apoptosis-related genes (Bcl-2, Bax and PUMA) which were previously identified as Slug targets. Bcl-2 expression was down-regulated in Slug-deficient cells. In conclusion, SFRP2 appears to interact with Slug to affect the apoptosis of hypertrophic scar fibroblasts.     

Activity of 25-Hydroxylase in Human Gingival Fibroblasts and Periodontal Ligament Cells

Background

We previously demonstrated that 25-hydroxyvitamin D₃ concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D₃ can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells.

Methodology/Principal Findings

Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D₃, human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D₃ that resulted in the production of 1α,25-dihydroxyvitamin D₃. Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D₃ synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1β and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells.

Conclusions

The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.     

Interleukin-6 negatively regulates Porphyromonas gingivalis fimbria-stimulated fibronectin expression in human gingival fibroblasts

Our previous study demonstrated that fibronectin (FN) is a negative regulator of Porphyromonas gingivalis fimbria-induced pathogenesis in the initiation and development of chronic periodontal diseases. We show herein the regulatory action of interleukin-6 (IL-6) on FN expression in fimbria-treated human gingival fibroblasts. Interestingly, the decrease in FN expression in the cells treated with fimbriae at a high dose (8 microg of protein ml(-1)) was negated by treatment with anti-IL-6 antibody. Also, the increase in FN expression in cells treated with fimbriae at a low dose (1 microg of protein ml(-1)) was inhibited by exogenous IL-6. These results suggest that P. gingivalis fimbria-stimulated FN expression in human gingival fibroblasts is negatively regulated by endogenous IL-6.     

Mechanical force inhibits osteoclastogenic potential of human periodontal ligament fibroblasts through OPG production and ERK‐mediated signaling

Periodontal ligament and gingival fibroblasts play important roles in bone remodeling. Periodontal ligament fibroblasts stimulate bone remodeling while gingival fibroblasts protect abnormal bone resorption. However, few studies had examined the differences in stimulation of osteoclast formation between the two fibroblast populations. The precise effect of mechanical forces on osteoclastogenesis of these populations is also unknown. This study revealed that more osteoclast‐like cells were induced in the co‐cultures of bone marrow cells with periodontal ligament than gingival fibroblasts, and this was considerably increased when anti‐osteoprotegerin (OPG) antibody was added to the co‐cultures. mRNA levels of receptor activator of nuclear factor‐kappaB ligand (RANKL) were increased in both populations when they were cultured with dexamethasone and vitamin D₃. Centrifugal forces inhibited osteoclastogenesis of both populations, and this was likely related to the force‐induced OPG up‐regulation. Inhibition of extracellular signal‐regulated kinase (ERK) signaling by a pharmacological inhibitor (10 µM PD98059) or by siERK transfection suppressed the force‐induced OPG up‐regulation along with the augmentation of osteoclast‐like cells that were decreased by the force. These results suggest that periodontal ligament fibroblasts are naturally better at osteoclast induction than gingival fibroblasts, and that centrifugal force inhibited osteoclastogenesis of the periodontal fibroblasts through OPG production and ERK activation. J. Cell. Biochem. 106: 1010–1019, 2009. © 2009 Wiley‐Liss, Inc.     

Cell survival and gene expression under compressive stress in a three-dimensional in vitro human periodontal ligament-like tissue model

This study investigated cell survival and gene expression under various compressive stress conditions mimicking orthodontic force by using a newly developed in vitro model of human periodontal ligament-like tissue (HPdLLT). The HPdLLT was developed by three-dimensional culturing of human periodontal ligament fibroblasts in a porous poly-l-lactide matrix with threefold increased culture media permeability due to hydrophilic modification. In vitro HPdLLTs in experimental groups were subjected to 5, 15, 25 and 35 g/cm² compressive stress for 1, 3, 7 or 14 days; controls were cultured over the same periods without compressive stress. Cell morphology and cell apoptosis in the experimental and control groups were investigated using scanning electron microscopy and caspase-3/7 detection. Real-time polymerase chain reaction was performed for seven osteogenic and osteoclastic genes. Similar extracellular matrix and spindle-shaped cells were observed inside or on the surface of in vitro HPdLLTs, with no relation to compressive stress duration or intensity. Similar caspase-3/7 activity indicating comparable apoptosis levels was observed in all samples. Receptor activator of nuclear factor kappa-B ligand and bone morphogenetic protein 2 genes showed characteristic “double-peak” expression at 15 and 35 g/cm² on day 14, and alkaline phosphatase and periodontal ligament-associated protein 1 expression peaked at 5 g/cm² on day 14; other genes also showed time-dependent and load-dependent expression patterns. The in vitro HPdLLT model system effectively mimicked the reaction and gene expression of the human periodontal ligament in response to orthodontic force. This work provides new information on the effects of compressive stress on human periodontal ligament tissue.     

miRNA-145通过下调ROCK1的表达抑制人牙周膜成纤维细胞的迁移

目的:研究微小核糖核酸-145(microRNA-145,miRNA-145)对人牙周膜成纤维细胞迁移的影响及其作用机制。方法:体外采用酶消化法培养人牙周膜成纤维细胞并传代,将其分为对照组和转染miRNA-145组,按50 ng/mL的miRNA-145浓度转染人牙周膜成纤维细胞,转染72 h后提取各组蛋白,用Western blot检测miRNA-145的靶蛋白ROCK1的表达水平的相关变化;采用划痕试验检测各组划痕细胞间距离的相关变化,选取划痕后的0 h、24 h、48 h、72 h时间点,测量各时间点划痕细胞间的距离并计算平均值。结果:与对照组相比,转染miRNA-145后,miRNA-145靶蛋白ROCK1的表达量显著降低(p0.05);转染24 h、48 h后细胞间距离的均值大于对照组(p0.05)。结论:miRNA-145可能通过下调ROCK1的表达抑制人牙周膜成纤维细胞的迁移。     

Interleukin-1 superfamily member,interleukin-33, in periodontal diseases

Interleukin (IL) -33 is a nuclear protein that is released from damaged cells and acts as an alarmin. We investigated the expression of IL-33 in human gingival fibroblasts after stimulation by tumor necrosis factor alpha (TNF-α). Human periodontal tissue samples were collected and fixed in phosphate-buffered 4% formalin in saline and processed to paraffin blocks. TNF-α was immunostained in samples of ten periodontitis patients and ten controls. Human gingival fibroblasts were isolated using an explant culture technique. The influence of TNF-α on IL-33 in gingival fibroblasts was analyzed using enzyme-linked immunosorbent assay (ELISA). The number of TNF-α positive cells was significantly greater in periodontitis samples than in controls. TNF-α was located mainly in macrophage- and fibroblast-like cells, vascular endothelial cells and epithelial cells. Analysis of IL-33 expression in cell culture lysates showed that TNF-α induced IL-33 in cultured gingival fibroblasts. Periodontitis samples are characterized by Th2 cell dominance, which has been linked to anti-inflammatory responses and periodontal repair. TNF-α-induced IL-33 may link inflammation directly to the IL-33-dependent stimulation of Th2 cytokine producing cells and participate in the induction of lymphocytes, which results in protective, anti-inflammatory and reparative responses.     

Isolation and characterization of cultured human periodental ligament fibroblast-specific cDNAs

The molecular mechanisms that control the function of periodontal ligament (PDL) fibroblasts remain unclear. We speculated that the character of differentiating PDL fibroblasts is defined by the altered expansion of specific genes not found in neighboring gingival fibroblasts in the periodontium. To expand this set, subtractive hybridization was applied between cultured human PDL and gingival fibroblasts to identify genes differentially expressed in PDL. Consequently five candidate clones, PDLs (periodontal ligament specific) 5, -17, -22, -25, and -31 were identified and characterized by homology search, Northern analysis, and in situ hybridization. Although the mRNAs of these clones were expressed by bone marrow cells and rarely by gingival fibroblasts, the highest expression was detected in the PDL cells, which were uniformly distributed throughout the whole PDL. Amongst the five candidate clones, we focused on PDLs17, because it is a hypothetical protein whose biological function has not been reported yet in the database. Polyclonal antiserum raised against PDLs17 peptide was made, and stained the PDL fibroblasts, osteoblast-like cells and stromal cells in the bone marrow, but not gingival fibroblasts. The results suggest that clones, PDLs5, -17, -22, -25, and -31 may be used as PDL fibroblast-specific markers, and that PDLs17 could act as an important factor in the differentiation process of PDL fibroblasts.     

糖尿病大鼠牙周膜成纤维细胞组织中TNF-α的表达及意义

目的：通过检测TNF-α在糖尿病大鼠牙周组织中的表达情况,探讨高糖环境下肿瘤坏死因子与口腔疾病发生发展的关系,为临床治疗提供理论依据。方法：选取SD大鼠40只,随机分为实验组（30只）和对照组（10只）。实验组采取腹腔注射链脲佐菌素的方法建立糖尿病大鼠模型,观察大鼠牙周组织的变化情况。采用RT-PCR法检测两组大鼠牙周膜成纤维细胞中TNF-α的表达水平,CCK-8法测定大鼠牙周膜成纤维细胞的增殖情况,分析TNF-α对PDLFs增殖的抑制作用。结果：实验组大鼠牙周组织胶原纤维束排列紊乱,有炎症细胞浸润;对照组大鼠牙龈未见红肿或出血。TNF-α在糖尿病大鼠牙周膜成纤维细胞中呈高表达,在健康大鼠牙周组织中不显著,差异具统计学意义（P〈0.05）。TNF-α可抑制PDLFs增殖,并且成浓度依赖性,随着浓度的增加,TNF-α对PDLFs增殖的抑制作用逐渐增强,差异具统计学意义（P〈0.05）。结论：糖尿病可增加牙周疾病的发病几率,糖尿病患者体内的TNF-α对牙周疾病的严重程度起重要作用。     

人牙龈成纤维细胞与牙周膜细胞的生物活性

采用组织块法分离培养牙周膜细胞和牙龈成纤维细胞,测定二者的增殖特性和ALP活性,利用免疫组化和FCM方法比较Ⅰ、Ⅲ型胶原、BMP的表达情况,以观察对比两种细胞的生物学特性的异同。找出牙龈成纤维细胞和牙周膜细胞在胶原基质合成方面存在差异,发现Ⅰ、Ⅲ型胶原可作为鉴别两种细胞的标志物,ALP与BMP可作为鉴别两种细胞的标志,牙周膜细胞比牙龈成纤维细胞具有较强的成骨能力。从而为今后改良两种细胞成为牙周组织工程的种子细胞奠定基础。