RANKL Gene Expression Research Articles

Prostaglandin E2 affects osteoblast biology in a dose-dependent manner: An in vitro study

ObjectiveThis study aimed to determine in vitro how exogenous PGE2 affects the expression of genes in cultured osteoblasts by relative quantitation PCR. DesignCultured osteoblasts were exposed to 10−3M, 10−5M or 10−7M PGE2 over 5, 10, 15 and 20 days. ResultsRANKL expression was higher after 5 days of exposure (p<0.05), but thereafter reduced in those treated with the two lower doses of PGE2 (p<0.01). RANKL/OPG ratio reported in favour of OPG gene expression and alkaline phosphatase gene expression increased in osteoblasts exposed to the two lower doses of the eicosanoid after 15 days. Conversely, prostaglandin E synthase, a cytokine produced during PGE2 synthesis, gene expression was significantly reduced at 15 and 20 days (p<0.01 and 0.05 respectively). The results from this study add to the current knowledge of the mechanisms by which PGE2 modulates the osteoblast biology in a dose-dependent manner. ConclusionsIt is proposed that PGE2at a low dose switch osteoblast's biology in favour of bone apposition by: first, inducing a significantly higher OPG gene expression overwhelming RANKL gene expression; second, reducing PGEs synthesis; and third, increasing ALP gene expression. An opposite effect is expected when the concentration of the eicosanoid overpass certain levels.

Modulation of gene expression in human subchondral bone cells co-cultured with human articular chondrocytes

Purpose: To investigate modulation effects in gene expression in a co-culture model of human bone cells and human condrocytes from osteoarthritic (OA) and normal (N) joint tissue. Methods: Human bone cells from the subchondral plate (hSBCs) and human articular chondrocytes (hCHs) were used. Cells were isolated from knee tissue discarded during joint replacement surgeries using standard procedures. For the co-culture experiments hCHs were cultured on the membrane of BD Falcon culture inserts while hSBCs were cultured in the wells of companion plates. In this way both cells types are co-cultured without physical contact but allowing paracrine interactions. Three different combinations were tested, namely, normal (N) hSBCs co-cultured with N hCHs, N hSBCs co-cultured with OA-hCHs and OA-hSBCs with OA-hCHs. After 7, 14 and 21 days of co-culture, total RNA was extracted using Trizol® following the manufacturer instructions. qRT-PCR analyses were performed to study the effects on gene expression in the hSBCs due to the co-culture with hCHs. In particular, we looked at the expression of alkaline phosphatase (ALP), osteocalcin (OCN), collagen type I (COLI), osteoprotegerin (OPG) and RANKL in the hSBCs. Results: hSBCs monocultures: Differences were found in gene expression between the N and OA hSBCs. ALP expression increased progressively from day 7 to day 21 in both normal and OA hSBCs monocultures up to about 4 fold with respect to the basal cells expression. OCN gene was similarly expressed in normal and OA cells in monoculture at 7 days, but its expression was markedly increased in the OA cells at both 14 and 21 days with respect to the normal cells (15.5 and 16 fold increase, compared to 4.5 and 2.8 fold increase, respectively). Conversely, COLI gene expression was reduced in the OA cells with respect to the normal cells (7.8 vs. 14.9) at 21 days of culture. Additionally, while in the N hSBCs OPG increased up to 22 fold at 14 d and was moderately reduced to 18 fold at 21 d, in the OA hSBCs, OPG increased only to 8 fold at 14 d and was further reduced to 5 fold at 21 d. Finally, both N and OA hSBCs RANKL gene expression was downregulated at 7, 14 and 21 d with respect to the basal cells expression. Co-cultures with normal or OA chondrocytes: ALP, OCN and COLI gene expression in both normal and OA hSBCs was reduced at 21 d when these cells were co-cultured with OA hCHs compared to the expression in N hSBCs co-cultured with N hCHs. OPG gene expression was higher in hSBCs co-cultured with OA hCHs at 14 d but this effect was inversed at 21 d (with respect to the co-culture with normal hCHs). In the case of RANKL, while in N hSBCs co-cultured with N hCHs expression was downregulated for all the time points investigated as was the case in the N hSBCs monoculture, in the hSBCs cultured with OA hCHs RANKL expression was increased in both N and OA cells at 14 d and in the OA hSBCs this effect was also seen at 21 d. Conclusions: Co-culture of hSBCs from either normal or OA tissue with hCHs from OA tissue modulated the expression of ALP, OCN, COLI, OPG and RANKL genes. These results suggest that paracrine signals from chondrocytes in OA might be capable of regulating gene expression of relevant osteoblastic genes involved in bone matrix formation and remodelling in both normal and OA hSBCs cells.

P3-01-12: Prognostic Impact of RANK, RANKL and OPG Gene Expression in ER Positive Primary Breast Cancer.

Abstract Background The cell surface receptor RANK (receptor activator of NFκB), its ligand (RANKL) and the decoy receptor of RANKL osteoprotegerin (OPG) play an important functional role in bone physiology and in bone metastasis by regulating osteoclasts. Just recently it was shown that tumor-infiltrating lymphocytes can stimulate breast cancer metastases through RANK-RANKL signalling. Material and methods: We analyzed gene expression of RANK, RANKL and OPG in a combined Affymetrix dataset of 307 ER positive breast cancers from our institutions which were either untreated of treated with chemotherapy. Kaplan Meier analysis of disease free survival and Cox regression analysis was applied to examine the prognostic value of the different markers. Results: We observed no significant difference in survival when samples were analyzed according to either RANK or RANKL mRNA expression. In contrast when samples were stratified in quartiles of OPG expression a positive linear relationship of survival with the expression of OPG was observed. Moreover since OPG demonstrated a bimodal type of expression a cutoff value can be derived from the expression data. Using this cutoff value a hazard ration of 2.14 (95% CI 1.27−3.61; P=0.004 for low OPG expression was detected. OPG expression correlated with lower proportion of grade 3 tumors (15.7% vs 27%; P=0.022) and a higher proportion of PgR positive samples (86.2% vs 71.4%; P=0.002). No significant differences were observed for lymph node status, age, tumor size and HER2 status. In multivariate analysis only lymph node status remained significant while OPG, Ki67, age, grade, and PgR only displayed a trend towards significance. Conclusion: Expression of osteoprotegerin seems to correlate with good prognosis in ER postive breast cancer. These data are in line with in vitro studies demonstration that OPG inhibits RANKL induced migration of tumor cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-12.

PTHrP increases RANKL expression by stromal cells from giant cell tumor of bone

Giant cell tumor of bone (GCT) presents with numerous osteoclast-like multinucleated giant cells that are principally responsible for the extensive bone resorption by the tumor. Although the precise etiology of GCT remains uncertain, the accumulation of giant cells is partially due to the high expression of the receptor activator of nuclear factor-κB ligand (RANKL) from the neoplastic stromal cells. Here, we have investigated whether parathyroid hormone-related protein (PTHrP) plays a role in the pathogenesis of GCT. Immunohistochemistry results revealed PTHrP expression in the stromal cells of the tumor, and that its receptor, the parathyroid hormone type 1 receptor (PTH1R), is expressed by both the stromal cells and giant cells. PCR and Western blot analyses confirmed the expression of PTHrP and PTH1R by isolated stromal cells from five patients presenting with GCT. Treatment of GCT stromal cells with varying concentrations of PTHrP (1-34) significantly increased both RANKL gene expression and the number of multinucleated cells formed from RAW 264.7 cells in co-culture experiments, whereas inhibition of PTHrP with a neutralizing antibody decreased RANKL gene expression. These results suggest that PTHrP is expressed within GCT by the stromal cells and can contribute to the abundant RANKL expression and giant cell formation within the tumor.

HRpn13, a newly identified component of the 19S particle, regulates proliferation, differentiation, and function in the human osteoblast-like cell line MG63

The 26S proteasome is a key component of the ubiquitin-proteasome system, a process responsible for the majority of cellular protein degradation. The function of the proteasomal ubiquitin receptor hRpn13, a component of the 26S proteasome, is not completely understood. To investigate the role of hRpn13 in the ubiquitin-proteasome system in osteoblasts, the effects of suppressing and overexpressing the hRpn13 gene on proliferation, differentiation, and function of human osteoblast-like MG63 cells were examined. After knockdown of hRpn13 by small interfering RNA, changes in osteoblast proliferation were evaluated by methyl-thiazolyl-tetrazolium assay. There was an increase in markers for osteoblast proliferation, specifically alkaline phosphatase activity, and elevated protein levels of osteocalcin, proliferating cell nuclear antigen (PCNA), and ubiquitin. Furthermore, hRpn13 knockdown also resulted in a decrease in the ratio between the gene expressions of RANKL and OPG, key players in the pathogenesis of bone diseases that influence the normal balance between bone formation and resorption. In contrast, overexpression of hRpn13 inhibited the proliferation of MG63 cells, and decreased alkaline phosphatase activity as well as protein levels of osteocalcin, PCNA, and ubiquitin while the ratio of RANKL to OPG expression increased. To confirm the function of hRpn13 in the ubiquitin-proteasome pathway, osteoblast proliferation enhancement and ubiquitin accumulation after hRpn2 knockdown was assessed. The results suggest that overexpression of hRpn13 negatively influences proliferation and osteogenic differentiation in MG63 cells. The evidence implies that hRpn13 modulates the influence of osteoblasts on osteoclasts by controlling the stability of regulatory proteins in osteoblasts. In summary, overexpression of hRpn13 promoted the activity of the ubiquitin-proteasome system.

Frontiers in vitamin D; basic research and clinical application. Is VDR a factor of bone volume regulator in bone tissues?

Vitamin D is one of the best established hormone to exert beneficial action for bone mass control and bone metabolism. Such vitamin D action for bone tissue is recapitulated in intact animals, while vitamin D is well known to potentiate bone resorption by inducing gene of RANKL an osteoclastic inducer. By genetic characterization of VDR in mice, VDR was found to serve as a negative regulator in intact bone. Moreover, RANKL gene expression level in intact bone was down-regulated in osteoblast-specific VDR KO mice. Thus, these findings indicate that VDR in bone tissue is a negative regulator for bone mass control.

A preliminary study on the FAM5C expression in generalized chronic periodontitis

The Family with sequence similarity 5 member C (FAM5C) has been suggested to contribute in aggressive periodontitis. However, there is no data regarding its role in chronic periodontitis. The aim of this study was to evaluate the FAM5C expression in chronic periodontitis and to study association of FAM5C with key immunoinflammatory markers. Gingival biopsies were harvested from periodontally healthy subjects (n = 10) and chronic periodontitis subjects (n = 15). The levels of mRNA of FAM5C, interleukin (IL)-17, IL-6, IL-23, IL-10, IL-4, interferon-c, toll-like receptor (TLR)-2, TLR-4, osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), tumor necrosis factor (TNF)-a, transforming growth factor-b, transcription factor forkhead box p3, and transcription factor orphan nuclear receptor C2 were evaluated by real-time polymerase chain reaction. FAM5C mRNA levels were not different between periodontally healthy and diseased tissues (P > 0.05). Gene expressions of IL-17, TNF-a, OPG, RANKL, TLR-2, and TLR-4 were higher in periodontitis, when compared to periodontally healthy sites (P < 0.05), while no differences between groups were observed for the other genes evaluated (P > 0.05). There were no correlations between the gene expression of FAM5C and the other immunoinflammatory markers (P > 0.05). Within the limits of this study, it seems that FAM5C expression does not contribute to chronic periodontitis.

Constitutive expression of cathepsin K in the human intervertebral disc: new insight into disc extracellular matrix remodeling via cathepsin K and receptor activator of nuclear factor-κB ligand

IntroductionCathepsin K is a recently discovered cysteine protease which cleaves the triple helical domains of type I to II collagen. It has been shown to be up-regulated in synovial tissue from osteoarthritic and rheumatoid patients, and is a component in normal and nonarthritic cartilage, where it increases with aging. Studies on heart valve development have recently shown that receptor activator of nuclear factor-κB ligand (RANKL) acts during valve remodeling to promote cathepsin K expression. Since extracellular matrix remodeling is a critical component of disc structure and biomechanical function, we hypothesized that cathepsin K and RANKL may be present in the human intervertebral disc.MethodsStudies were performed following approval of the authors' Human Subjects Institutional Review Board. Six annulus specimens from healthier Thompson grade I to II discs, and 12 specimens from more degenerate grade III to IV discs were utilized in microarray analysis of RANKL and cathepsin K gene expression. Immunohistochemistry was also performed on 15 additional disc specimens to assess the presence of RANKL and cathepsin K.ResultsCathepsin K gene expression was significantly greater in more degenerated grade III to IV discs compared to healthier grade I to II discs (P = 0.001). RANKL was also identified with immunohistochemistry and molecular analyses. RANKL gene expression was also significantly greater in more degenerated discs compared to healthier ones (P = 0.0001). A significant linear positive correlation was identified between expression of cathepsin K and RANKL (r2 = 92.2; P < 0.0001).ConclusionsExtracellular matrix remodeling is a key element of disc biology. Our use of an appropriate antibody and gene expression studies showed that cathepsin K is indeed present in the human intervertebral disc. Immunolocalization and molecular analyses also confirmed that RANKL is present in the human disc. Expression of RANKL was found to be significantly greater in more degenerated compared to healthier discs (P = 0.0001). Cathepsin K gene expression levels showed a positive, significant correlation with RANKL expression. Based on these data, we propose that cathepsin K plays a significant role in disc matrix remodeling and in matrix degradation in the proinflammatory cytokine-rich microenvironment of the degenerating disc.

Strontium ranelate inhibits key factors affecting bone remodeling in human osteoarthritic subchondral bone osteoblasts

In osteoarthritis (OA) the progression of cartilage degeneration has been associated with remodeling of the subchondral bone. Human OA subchondral bone osteoblasts were shown to have an abnormal phenotype and altered metabolism leading to an abnormal resorptive process. Bone resorption is suggested to occur, at least in part, through the increased levels of two proteolytic enzymes, MMP-2 and MMP-9, and RANKL, which are mainly produced by osteoblasts. In this study, we investigated in human OA subchondral bone osteoblasts the modulatory effect of strontium ranelate on the above key factors. Human subchondral bone osteoblasts were cultured in a medium containing 0.1, 1 and 2 mM of strontium ranelate for 18 h for mRNA and 72 h for protein determination. The effect of strontium ranelate was evaluated on the expression (qPCR) of MMP-2, MMP-9, OPG, RANKL (total), RANKL-1, and RANKL-3, on the production of OPG (ELISA), membranous RANKL (flow cytometry), and MT1-MMP, ADAM17, and ADAM19 (Western blot). After incubation of osteoblasts with pre-osteoclasts (i.e., differentiated human peripheral blood mononuclear cells), the resorbed surface was measured using a sub-micron synthetic calcium phosphate thin film. Firstly, the expression levels of MMP-2, MMP-9, OPG, and RANKL were determined in normal and OA subchondral bone osteoblasts. As expected, the gene expression of MMP-9 and RANKL were not detectable in normal cells, whereas MMP-2 was very low but detectable and OPG demonstrated high gene expression. Further experiments looking at the effect of strontium ranelate on expression levels, except for OPG, were performed only on the OA subchondral bone osteoblasts. In OA cells, the expression levels of MMP-2 and MMP-9 were significantly decreased by strontium ranelate at 1mM (p≤0.005, p≤0.02, respectively) and 2 mM (p≤0.003, p≤0.007), and for MMP-9 only at 0.1 mM (p≤0.05). In normal cells, the expression of OPG was increased with strontium ranelate at 2 mM, and in OA both the expression (p≤0.02) and synthesis (p≤0.002) of OPG were significantly increased with strontium ranelate at 1 and 2 mM. RANKL (total) as well as the isoforms RANKL-1 and RANKL-3 were significantly increased by strontium ranelate at 1 and 2 mM. Of note, it is known that the different RANKL isoforms differentially regulate RANKL membranous localization: RANKL-3, in contrast to RANKL-1, prevents such membranous localization. This is reflected by the significant (p≤0.02) reduction in the level of membranous RANKL by strontium ranelate at 2 mM. This latter finding was not likely to be related to a proteolytic cleavage of membranous RANKL, as the enzymes known to cleave it, MT1-MMP, ADAM17 and ADAM19, were unaffected by strontium ranelate. In addition, OA osteoblasts treated with strontium ranelate induced a significant (p≤0.002) decrease in resorbed surface at the three tested concentrations. This study provides new insights into the mode of action of strontium ranelate on the metabolism of human OA subchondral bone osteoblasts. These data suggest that strontium ranelate may exert a positive effect on OA pathophysiology by inhibiting, in these cells, the synthesis of key factors leading to bone resorption, a feature associated with the OA process.

Coptisine inhibits RANKL-induced NF-κB phosphorylation in osteoclast precursors and suppresses function through the regulation of RANKL and OPG gene expression in osteoblastic cells

Excessive receptor activator of NF-κB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. The downregulation of RANKL expression and its downstream signals may be an effective therapeutic approach to the treatment of bone loss diseases such as osteoporosis. Here, we found that coptisine, one of the isoquinoline alkaloids from Coptidis Rhizoma, exhibited inhibitory effects on osteoclastogenesis in vitro. Although coptisine has been studied for its antipyretic, antiphotooxidative, dampness dispelling, antidote, antinociceptive, and anti-inflammatory activities in vitro and in vivo, its effects on osteoclastogenesis have not been investigated. Therefore, we evaluated the effects of coptisine on osteoblastic cells as well as osteoclast precursors for osteoclastogenesis in vitro. The addition of coptisine to cocultures of mouse bone marrow cells and primary osteoblastic cells with 10(-8) M 1α,25(OH)(2)D(3) caused significant inhibition of osteoclast formation in a dose-dependent manner. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that coptisine inhibited RANKL gene expression and stimulated the osteoprotegerin gene expression induced by 1α,25(OH)(2)D(3) in osteoblastic cells. Coptisine strongly inhibited RANKL-induced osteoclast formation when added during the early stage of bone marrow macrophage (BMM) cultures, suggesting that it acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, coptisine inhibited NF-κB p65 phosphorylations, which are regulated in response to RANKL in BMMs. Coptisine also inhibited the RANKL-induced expression of NFATc1, which is a key transcription factor. In addition, 10 μM coptisine significantly inhibited both the survival of mature osteoclasts and their pit-forming activity in cocultures. Thus, coptisine has potential for the treatment or prevention of several bone diseases characterized by excessive bone destruction.

The RANKL–OPG system is differentially regulated by supragingival and subgingival biofilm supernatants

Periodontitis is an inflammatory condition that destroys the tooth supporting tissues, including the alveolar bone. It is triggered by polymicrobial biofilms attaching on tooth surfaces, which can be supragingival or subgingival. Bone resorption is triggered by receptor activator of NF-κB ligand (RANKL) and blocked by its soluble decoy receptor osteoprotegerin (OPG), which are cytokines of the tumor necrosis factor ligand and receptor families, respectively. The present study aimed to comparatively investigate the effects of the Zürich in vitro supragingival and subgingival biofilm models, on RANKL and OPG gene expression in primary human gingival fibroblasts (GF) cultures. The cells were challenged with biofilm culture supernatants for up-to 24 h. RANKL and OPG gene expression in the cells was analyzed by quantitative real-time polymerase chain reaction (qPCR) and their relative RANKL/OPG ratio was calculated. Both biofilm supernatants induced RANKL expression, but the subgingival caused a more pronounced up-regulation compared to the supragingival (10-fold at 6 h and 100-fold at 24 h). Changes in OPG expression in response to either biofilm were more limited. Accordingly, the subgingival biofilm caused a greater enhancement of the relative RANKL/OPG ratio (4-fold at 6 h and 110-fold 24 h). In conclusion, subgingival biofilms exhibit a stronger potency for inducing molecular mechanisms of bone resorption than supragingival biofilms, in line with their higher virulence nature for the development of periodontitis.

Patterns of osteoprotegerin or nuclear factor kappa B ligand gene expression in the treatment of bone defect with bone marrow stem cell transplantation and low-frequency vibration

To explore the effect of low frequency vibration (LFV)on the osteogenic differentiation regulating capability of bone marrow stromal cell (BMSC)and the expressions of OPG (osteoprotegerin) mRNA and RANKL (nuclear factor kappa B ligand) mRNA through living animal experiment. Both BMSC transplantation and low-frequency vibration were employed to treat bone defects. The groups were randomized into non-vibration and vibration of different frequencies. The vibration group received vibrating interventions at Day 7 for 5 weeks. After vibrations, the BMSC OPG and RANKL mRNA of different frequency groups were detected. The BMSC OPG and RANKL gene expressions significantly increased (P < 0.05), especially at 25 Hz (P < 0.01). And for the vibration group at 100 Hz, the BMSC OPG and RANKL gene expressions decreased (P < 0.05). Low-frequency vibration may promote the osteogenic differentiation capability of BMSC probably through regulating the OPG/RANKL mRNA expression, directly promoting bone formation and inhibiting bone resorption.

Trabecular bone microstructure and local gene expression in iliac crest biopsies of men with idiopathic osteoporosis

Male idiopathic osteoporosis (MIO) is a metabolic bone disease that is characterized by low bone mass, microstructural alterations, and increased fracture risk in otherwise healthy men. Although the detailed pathophysiology of MIO has yet to be clarified, evidence increasingly suggests an osteoblastic defect as the underlying cause. In this study we tested the hypothesis that the expression profile of certain osteoblastic or osteoblast-related genes (ie, WNT10B, RUNX2, Osterix, Osteocalcin, SOST, RANKL, and OPG) is different in iliac crest biopsies of MIO patients when compared with healthy controls. Furthermore, we investigated the relation of local gene expression characteristics with histomorphometric, microstructural, and clinical features. Following written informed consent and diligent clinical patient characterization, iliac crest biopsies were performed in nine men. While RNA extraction, reverse-transcription, and real-time polymerase chain reactions (PCRs) were performed on one biopsy, a second biopsy of each patient was submitted for histomorphometry and micro-computed tomography (µCT). Age-matched bone samples from forensic autopsies served as controls. MIO patients displayed significantly reduced WNT10B, RUNX2, RANKL, and SOST expression. Performing µCT for the first time in MIO biopsies, we found significant decreases in trabecular number and connectivity density. Trabecular separation was increased significantly, but trabecular thickness was similar in both groups. Histomorphometry revealed decreased BV/TV and osteoid volume and fewer osteoclasts in MIO. By providing evidence for reduced local WNT10B, RUNX2, and RANKL gene expression and histomorphometric low turnover, our data support the osteoblast dysfunction model discussed for MIO. Further, MIO seems to lead to a different microstructural pathology than age-related bone loss.

The Role of Osteoprotegrin/Nuclear Factor kB Ligand in Coronary Collateral Development

Osteoprotegrin (OPG), as a member of the TNF family is demonstrated to be a potential regulator of the endothelial function and angiogenesis by neutralization of nuclear factor kB ligand (RANKL). We investigated the OPG and RANKL gene expressions in circulating peripheral blood mononuclear cells (PBMCs) of Coronary artery disease (CAD) patients with different extents of coronary collateral development. In a cross-sectional study, 206 individuals with angiographically documented CAD were recruited. Severity of CAD was defined by the number of involved coronary vessels. The Rentrop scoring system was used to grade the extent of collateral development. Grade 0 or 1 collateralization was considered poor collateralization. RNA extraction and cDNA synthesis were performed. OPG and RANKL gene expressions were evaluated using quantitative real-time PCR. Among patients with CAD, 48.5% (100), 16.5%(34) and 35%(72) were considered to have one to three degrees of coronary artery involvement, respectively. The OPG and the ratio of OPG to RANKL expression were significantly elevated in patients with well-developed collateralization. In a logistic regression, severity of CAD was associated with a better collateral development, and OPG gene over expression was correlated with a better collateralization, independently of other variables. In conclusion, it seems that OPG might have an important role in prognosis of CAD; its up-regulation is parallel with CAD severity while it can enhance collateral development.

Gene expression of transcription factor NFATc1 in periodontal diseases

Periodontitis is a disease of infectious aetiology that causes inflammatory destruction of the tooth-supporting tissues. Activated T cells are central to the pathogenesis of the disease, by producing receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) that stimulates bone resorption. Antigenic activation of T cells is regulated by the induction of transcription factor nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). There is as yet no information on the potential involvement of NFATc1 in periodontal diseases. This study aimed to investigate NFATc1 gene expression levels in periodontal diseases, and analyse the potential correlation with RANKL expression and clinical periodontal parameters. In this cross-sectional study, gingival tissue biopsies were obtained from healthy (n = 10) and periodontally diseased (n = 58) sites. NFATc1 and RANKL gene expression levels in these samples were analysed by quantitative real-time polymerase chain reaction. Compared with healthy subjects, patients with gingivitis, chronic and aggressive periodontitis, exhibited higher NFATc1 expression, which proved to be statistically significant in the periodontitis groups. NFATc1 and RANKL expression levels strongly correlated with each other, and with clinical periodontal parameters. The increased expression of NFATc1 in periodontitis denotes a role for this transcription factor in the pathogenesis of the disease.

Inhibition of NADPH Oxidases Prevents Chronic Ethanol-Induced Bone Loss in Female Rats

Previous in vitro data suggest that ethanol (EtOH) activates NADPH oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption observed in vivo after EtOH exposure. In a rat model in which cycling females were infused intragastrically with EtOH-containing liquid diets, EtOH significantly decreased bone formation and stimulated osteoblast-dependent osteoclast differentiation. These effects were reversed by exogenous 17-β-estradiol coadministration. Moreover, coadministration of N-acetyl cysteine (NAC), an antioxidant, or diphenylene iodonium (DPI), a specific Nox inhibitor, also abolished chronic EtOH-associated bone loss. EtOH treatment up-regulated mRNA levels of Nox1, 2, 4, and the receptor activator of nuclear factor-κB ligand (RANKL), an essential factor for differentiation of osteoclasts in bone. Protein levels of Nox4, a major Nox isoform expressed in nonphagocytic cells, was also up-regulated by EtOH in bone. 17-β-Estradiol, NAC, and DPI were able to normalize EtOH-induced up-regulation of Nox and RANKL. In vitro experiments demonstrated that EtOH directly up-regulated Nox expression in osteoblasts. Pretreatment of osteoblasts with DPI eliminated EtOH-induced RANKL promoter activity. Furthermore, EtOH induced RANKL gene expression, and RANKL promoter activation in osteoblasts was ROS-dependent. These data suggest that inhibition of Nox expression and activity may be critical for prevention of chronic EtOH-induced osteoblast-dependent bone loss.

Oral biofilm challenge regulates the RANKL-OPG system in periodontal ligament and dental pulp cells

Inflammatory bone destruction triggered by oral bacteria is a hallmark of chronic and apical periodontitis. Receptor activator of NF-κB ligand (RANKL) activates bone resorption, whereas osteoprotegerin (OPG) blocks its action. These are members of the tumor necrosis factor ligand and receptor families, respectively. Although individual oral pathogens are known to regulate RANKL and OPG expression in cells of relevance to the respective diseases, such as periodontal ligament (PDL) and dental pulp (DP) cells, the effect of polymicrobial oral biofilms is not known. This study aimed to investigate the effect of the Zürich in vitro supragingival biofilm model on RANKL and OPG gene expression, in human PDL and DP cell cultures, by quantitative real-time polymerase chain reaction. RANKL expression was more pronouncedly up-regulated in DP than PDL cells (4-fold greater), whereas OPG was up-regulated to a similar extent. The RANKL/OPG ratio was increased only in DP cells, indicating an enhanced capacity for inducing bone resorption. The expression of pro-inflammatory cytokine interleukin-1β was also increased in DP, but not PDL cells. Collectively, the high responsiveness of DP, but not PDL cells to the supragingival biofilm challenge could constitute a putative pathogenic mechanism for apical periodontitis, which may not crucial for chronic periodontitis.

Chemical modification of an implant surface increases osteogenesis and simultaneously reduces osteoclastogenesis: an in vitro study

The present study investigated the effect of a chemical modification of the SLA surface (SLActive surface) on human periodontal ligament (hPDL) cell (1) adhesion, (2) proliferation, (3) osteogenic differentiation (core binding factor α-1 [Cbfa-1], bone morphogenetic protein-7 [BMP-7] gene expression and alkaline phosphatase [ALP] activity) and (4) osteoclast formation and activity (osteoprotegerin [OPG] and receptor activator of nuclear factor-κ B ligand [RANKL] gene expression). The above activities were based on the hypothesis that the expression of such molecules might be dependent on the characteristics of the implant surface. hPDL cells were isolated and characterized for their mesenchymal origin, fibroblastic and osteoblastic phenotype. hPDL cells were cultured on smooth, SLA and SLActive implant surfaces (chemically modified). Cell attachment and proliferation were assessed for 5, 24, 72 h, 5 and 7 days. Cbfa-1, BMP-7, OPG and RANKL gene expression was assessed by RT-PCR and a colorimetric assay for ALP activity was applied. hPDL cells grown on SLActive surfaces demonstrated increased proliferation rates (24 h, 5 and 7 days of the incubation period), and ALP activity was found to be significantly upregulated (5, 72 h and 7 days) as compared with the SLA surfaces. After 7 days of culture, the gene expression of BMP-7, Cbfa-1 and OPG by hPDL cells was significantly upregulated, while RANKL gene expression was significantly downregulated in response to the SLActive surface. Chemical modification of a previously roughened implant surface increases hPDL proliferation and upregulates osteoblastic differentiation. It can also suppress osteoclastogenesis regulating the RANKL-RANK-OPG axis. Hence, an osteoprotective microenvironment is created around chemically modified implants that may enhance osseointegration.

Influence of bisphosphonates on the osteoblast RANKL and OPG gene expression in vitro

Bisphosphonates are widely used in the clinical treatment of bone diseases with increased bone resorption. In terms of side effects, they are widely known to be associated with osteonecrosis of the jaw (BONJ). The objective of this study was to evaluate the effect of bisphosphonates on the gene expression of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in vitro. Nitrogen-containing and non-nitrogen containing bisphosphonates have been compared. Human osteoblasts were stimulated with zoledronate and ibandronate at concentrations of 5 × 10(-5) M, 5 × 10(-6) M, and 5 × 10(-7) M over the experimental period of 14 days. Furthermore, the hOB cell lines were stimulated by clodronate at concentrations of 5 × 10(-3) M, 5 × 10(-5) M, and 5 × 10(-6) M. At each point in time, the gene expression levels of RANKL and OPG were quantified by real-time RT-PCR. The results showed a moderate enhancement of OPG gene expression whereas RANKL gene expression was strongly increased by nitrogen-containing bisphosphonates reaching a maximum after 14 days at high concentrations of 5 × 10(-5) M. Lower concentrations did not enhance the RANKL and OPG expression considerably. The non-nitrogen-containing bisphosphonate clodronate, however, effected OPG and RANKL gene expression much less, even at higher concentrations of 5 × 10(-3) M. The above-mentioned data suggest an enhanced RANKL/OPG gene expression after stimulation by bisphosphonates. Interestingly, clodronate might have little influence on osteoblast/osteoclast interaction with respect to OPG and RANKL gene expression.

Decreased RUNX2, RANKL and sost gene expression in transiliac bone biopsies of men with untreated idiopathic osteoporosis

Decreased RUNX2, RANKL and sost gene expression in transiliac bone biopsies of men with untreated idiopathic osteoporosis