Receptor Activator Of NF-kappaB Ligand Gene Research Articles

Enamel matrix derivative neutralized the effect of lipopolysaccharide on osteoprotegerin and receptor activator of nuclear factor kappa B ligand expression of osteoblasts

Objective In this study, we investigated the effects of enamel matrix derivative (EMD) on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) expression of osteoblasts in the presence of lipopolysaccharide (LPS). Study design OPG and RANKL gene expression and protein synthesis of MC3T3-E1 osteoblastic cells were examined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Results LPS inhibited OPG gene expression and protein synthesis, and stimulated RANKL gene expression and soluble RANKL synthesis. EMD enhanced OPG gene expression and protein synthesis, and inhibited RANKL gene expression and soluble RANKL synthesis. Furthermore, EMD neutralized the effects of LPS on OPG and RANKL expression in osteoblasts. Conclusions EMD might regulate the function of osteoblasts by elevating the ratio of OPG/RANKL gene expression, which is downregulated by LPS, and suppress the induction of osteoclastogenesis. Thereby, EMD might contribute to periodontal tissue regeneration.

Effects of calcitriol on the expression of vitamin D receptor, RANKL and osteoprotegerin in human periodontal ligament cells

To study the effects of 1, 25-dihydroxyvitamin D(3) (VD(3)) on the expression of vitamin D receptor (VDR), receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) in human periodontal ligament cells (hPDLC) populations and to analyze the potential mechanisms. Twelve hPDLC populations were primarily established from 12 donors individually. Two samples of each hPDLC population of passage three were treated respectively with 10(-8) mol/L VD(3) (V D(3) group) or 0.1% absolute ethyl alcohol as controls (control group). Six days later, the mRNA expression levels of VDR, RANKL and OPG in the samples were determined with real-time quantitative RT-PCR. The DNA base sequences upstream to the transcription start site of RANKL gene were also analyzed. Compared with the control group, the mRNA expression level of VDR increased significantly in the VD(3) group (P = 0.003), averagely (3.04 +/- 1.06) times of that in the control group; the mRNA expression level of RANKL was also up-regulated by VD(3) (P = 0.001), 9.82 (0.75-119.18) times of that in the control group; the OPG expression level was (94.48 +/- 39.15)% of the controls (P = 0.136); OPG/RANKL ratio was down-regulated in the VD(3) group to averagely 10.36% (1.01%-138.00%) of the controls (P = 0.003). No mutation was found in the DNA fragments upstream to the transcription start site in the RANKL gene and the genotypes of the polymorphism at -1832 (rs7984870, C/G) were not shown to be significantly related to the RANKL mRNA expression level. In hPDLC, VD(3) can significantly increase the mRNA expression level of VDR; VD(3) can increase RANKL mRNA expression level to decrease OPG/RANKL ratio, but it has little effect on OPG mRNA expression. The big differences of the RANKL mRNA regulation in response to VD(3) treatment among hPDLC populations may not be associated with the DNA sequences upstream to the transcription start site in the RANKL gene.

Genome-Wide Linkage Screen of Bone Mineral Density (BMD) in European Pedigrees Ascertained through a Male Relative with Low BMD Values: Evidence for Quantitative Trait Loci on 17q21–23, 11q12–13, 13q12–14, and 22q11

Bone mass is under strong genetic control, with heritability estimates greater than 50% and is likely determined by complex interactions between genetic and environmental factors. The objective of the study was to localize genes contributing to bone mineral density (BMD) variation. An autosomal genome-wide scan for BMD at the lumbar spine and femoral neck was conducted with variance components linkage methods. A total of 103 pedigrees (Network in Europe on Male Osteoporosis Family Study) ascertained through a male relative with low (Z-score < or = -2) BMD values at either lumbar spine or femoral neck. Nonparametric multipoint logarithm of the odds ratio scores for lumbar spine and femoral neck BMD values adjusted for age, gender, and body mass index. We identified a total of eight chromosomal regions with logarithm of the odds ratio score of 1.5 or greater (P < or = 5 x 10(-3)): on 1q42-43, 11q12-13, 12q23-24, 17q21-23, 21q22, and 22q11 for lumbar spine and on 5q31-33 and 13q12-14 for femoral neck BMD. Four of our detected quantitative trait loci (QTL) reached the genome-wide criteria for significant (17q,21-23, P < or = 2 x 10(-5)) or suggestive (11q12-13, 22q11, and 13q12-14, P < or = 7 x 10(-4)) linkage. Apart from 22q11, which is a novel QTL, all other loci provide consistent replication for previously reported QTLs for BMD and other bone-related traits. Finally, several of our specific-linkage areas encompass prominent candidate genes: type 1 collagen (COL1A1) and the sclerosteosis/van Buchem disease (SOST) genes on 17q21-23; the low-density lipoprotein receptor-related protein 5 (LRP5) gene on 11q12-13; and the rank ligand gene on 13q12-14. Further analysis of these positive regions by fine linkage disequilibrium mapping is thus warranted.

Transcriptional regulation of human RANK ligand gene expression by E2F1

Receptor activator of nuclear factor kappa B ligand (RANKL) is a critical osteoclastogenic factor involved in the regulation of bone resorption, immune function, the development of mammary gland and cardiovascular system. To understand the transcriptional regulation of RANKL, we amplified and characterized a 1890bp 5′-flanking sequence of human RANKL gene (−1782bp to +108bp relative to the transcription start site). Using a series of deletion mutations of the 1890bp RANKL promoter, we identified a 72bp region (−172 to −100bp) mediating RANKL basal transcriptional activity. Sequence analysis revealed a putative E2F binding site within this 72bp region in the human RANKL promoter. Overexpression of E2F1 increased RANKL promoter activity, while down-regulation of E2F1 expression by small interfering RNA decreased RANKL promoter activity. RT-PCR and enzyme linked immunosorbent assays (ELISA) further demonstrated that E2F1 induced the expression of RANKL. Electrophoretic gel mobility shift assays (EMSA) and antibody competition assays confirmed that E2F1 proteins bind to the consensus E2F binding site in the RANKL promoter. Mutation of the E2F consensus binding site in the RANKL promoter profoundly reduced the basal promoter activity and abolished the transcriptional modulation of RANKL by E2F1. These results suggest that E2F1 plays an important role in regulating RANKL transcription through binding to the E2F consensus binding site.

An Enhancer 20 Kilobases Upstream of the Human Receptor Activator of Nuclear Factor-κB Ligand Gene Mediates Dominant Activation by 1,25-Dihydroxyvitamin D3

Receptor activator of nuclear factor-kappaB ligand (RANKL) is a TNF-like factor that is both produced by osteoblasts, mesenchymal cells, and activated T cells and required for osteoclast maturation and survival. The gene is up-regulated by the two primary calcemic hormones, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and PTH. Previous studies have indicated that five enhancer regions located significantly upstream of the mouse Rankl transcriptional start site mediate up-regulation by 1,25(OH)2D3 and PTH. The most distal of these, termed mRLD5, is highly conserved in the human gene at -96 kb where it was also shown to be functionally active. Four additional mouse Rankl upstream enhancers are also highly conserved in the human gene at -20, -25, -75, and -87 kb. In the present studies, we characterized the activity of these regions, explored their capacity to mediate the actions of 1,25(OH)2D3, and identified the vitamin D response elements contained within the two most proximal segments. Interestingly, whereas the most distal of the five enhancers is the dominant mediator of 1,25(OH)2D3 activity in the mouse Rankl gene, that role in the human gene is manifested by the most proximal element at -20 kb. Importantly, activity at this region in response to 1,25(OH)2D3 was associated with a significant increase in histone acetylation as well as the enhanced recruitment of RNA polymerase II. Both likely reflect the primary role of this enhancer in human RANKL gene expression. Our studies confirm the complex nature of RANKL regulation and indicate that although the five enhancers are evolutionarily conserved across several species, their relative contributions to RANKL expression in response to 1,25(OH)2D3 may be different.

DACH1 negatively regulates the human RANK ligand gene expression in stromal/preosteoblast cells

Receptor activator of NF-kappaB ligand (RANKL) is a critical osteoclastogenic factor that is expressed on bone marrow stromal/preosteoblast cells. Most bone resorption stimuli induce osteoclast formation by modulating RANKL expression in these cells. However, little is known about the mechanisms regulating RANKL gene expression. We recently reported that heat shock factor-2 (HSF-2) is a downstream target for FGF-2 signaling to enhance RANKL gene transcription in marrow stromal/preosteoblast cells. In this study, we show that DACH1 (human homologue of Drosophila dachshund gene) negatively regulates RANKL gene expression and suppresses FGF-2-enhanced RANKL gene expression in these cells. DACH1 contains a conserved dachshund domain (DS) in the N-terminal region, which interacts with the nuclear co-repressor (NCoR) to repress gene expression. Co-expression of DACH1 with hRANKL promoter-luciferase reporter plasmid in normal human bone marrow-derived stromal cells significantly decreased (3.3-fold) FGF-2-stimulated hRANKL gene promoter activity. Deletion of DS domain abolished DACH1 inhibition of FGF-2-enhanced RANKL gene promoter activity. Western blot analysis confirmed that DACH1 suppressed FGF-2-stimulated RANKL expression in marrow stromal/preosteoblast cells. We show HSF-2 co-immune precipitated with DACH1 and that FGF-2 stimulation significantly increased (2.7-fold) HSF-2 binding to DACH1. Confocal microscopy analysis further demonstrated that FGF-2 promotes HSF-2 nuclear transport and co-localization with DACH1 in marrow stromal cells. Co-expression of NCoR with DACH1 significantly decreased (5.3-fold) and siRNA suppression of NCoR in DACH1 co-transfected cells increased (3.6-fold) RANKL promoter activity. Furthermore, DACH1 co-expression with NCoR significantly decreased (7.5-fold) RANKL mRNA expression in marrow stromal cells. Collectively, these studies indicate that NCoR participates in DACH1 repression of RANKL gene expression in marrow stromal/preosteoblast cells. Thus, DACH1 plays an important role in negative regulation of RANKL gene expression in marrow stromal/preosteoblast cells in the bone microenvironment.

Dexamethasone promotes osteoclastogenesis by inhibiting osteoprotegerin through multiple levels

Increased bone fragility attributed to osteopenia is a serious side effect of glucocorticoid treatment. Glucocorticoid-induced bone loss is caused primarily by hypofunction and apoptosis of osteoblasts, and secondarily by accelerated bone resorption. To explore the mechanism whereby dexamethasone (Dex) stimulates osteoclastogenesis in the coculture system, we analyzed the effect of Dex on the expression of both mouse osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL). Dex reduced OPG transcripts and OPG protein secretion by the ST2 osteoblastic cells. Since mainly the c-Jun homodimer maintains the steady-state transcription of the OPG gene, we examined the effect of Dex on c-Jun signaling in ST2 cells. Western blotting disclosed that Dex decreased the amount of phospho-c-Jun protein (p-c-Jun) and, correspondingly, the amount of the phosphorylated p46 isoform of Jun N-terminal kinase (JNK). The amount of phospho-SEK1 also decreased after Dex treatment, while the amounts of phospho-ERK and p38 remained constant. Among mitogen-activated protein (MAP) kinase inhibitors, the JNK inhibitor mimicked the inhibitory effect of Dex on OPG promoter activity. On the other hand, Dex treatment per se showed a nominal increase of RANKL gene expression. A part of Dex-mediated OPG gene suppression was achieved by the suppression of beta-catenin signaling. We speculate therefore that the bone resorptive action of Dex is mediated mainly by the inhibition of OPG by transrepressing the OPG gene through the AP-1 site, with a reduction (mediated mainly by the decrease in the p46 isoform of JNK) in the proportion of p-c-Jun in a JNK-dependent manner.

RANK ligand

RANK ligand (RANKL), a key mediator of bone resorption in normal and pathological states, is expressed as membrane-bound or soluble forms by tissues as diverse as lymph nodes, spleen, thymus and bone-forming cells. In normal bone turnover and in bone metastasis, RANKL stimulates the formation and activity of bone-removing cells, osteoclasts, by binding to its cognate receptor, RANK, on osteoclasts and their progenitors; these processes are disrupted by binding of RANKL to osteoprotegerin (OPG), a soluble decoy receptor. Whilst no mutations in the RANKL gene have yet been identified in human disease, mutations that result in enhanced RANK signalling through inactivation of OPG or activation of RANK are associated with Juvenile Paget's disease and familial expansile osteolysis, respectively. This review focuses on the central role of RANKL in bone resorption and on the therapeutic targeting of RANKL in osteoporosis, humoral hypercalcaemia of malignancy and bone metastasis.

Methylation Status of a Single CpG Locus 3 Bases Upstream of TATA-Box of Receptor Activator of Nuclear Factor-κB Ligand (RANKL) Gene Promoter Modulates Cell- and Tissue-Specific RANKL Expression and Osteoclastogenesis

Receptor activator of nuclear factor-kappaB ligand (RANKL) expression is tissue specific and limited to certain subsets of T-lymphocytes and stromal/osteoblastic cells. Even among osteoblasts, RANKL is expressed on about 20% of osteoblasts of the normal mouse. To clarify the mechanism of population-specific RANKL expression, we analyzed the effect of CpG methylation on its transcription, mRNA and protein expression as well as on osteoclastogenesis. Subpopulations of ST2 cells were used: P9, which expresses RANKL and supports osteoclastogenesis, and P16, which does not. By sodium bisulfite mapping, the rate of CpG methylation of the -65/+350 region, especially of CpG locus no. 1 three bases upstream of the TATA-box, was higher in P16 than in P9 ST2 cells. ChIP and gel shift assay showed that methylated CpG locus no. 1 was a target of MeCP2 binding that, in turn, blocked the binding of the TATA-box binding protein to the TATA-box. In vitro methylation by SssI of the promoter construct reduced its transcriptional activity at the steady state and its response to 1alpha,25(OH)2 vitamin D3. Conversely, treatment with DNA methylase inhibitor, 5-aza-2'-deoxycytidine, significantly restored RANKL expression and osteoclastogenesis in P16 cells. Except for primary cultured osteoblasts, CpG locus no. 1 was frequently methylated in various normal mouse tissues. We propose that the methylation status of the CpG locus three bases upstream of the TATA-box modulates the control of cell- and tissue-specific expression of RANKL gene and osteoclastogenesis. The heterogeneity of stromal/ osteoblastic cells in response to bone-resorbing stimuli may be attributed, in part, to the methylation status of the RANKL gene promoter.

Modulation of mouse RANKL gene expression by Runx2 and PKA pathway

Runx2 regulates the target genes characteristic of osteoblastic phenotypes, while exerting diverse and sometimes controversial effects on osteoblastic cells depending on their differentiation stage. Receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) is a membrane bound cytokine essential for osteo(chondro)clastogenesis. During endochondral ossification, while Runx2-positive hypertrophic chondrocytes express RANKL, the steady-state expression of the RANKL gene in osteoblastic cells is, at later stages, kept at a relatively low level to sustain the established bone. The aim of this study was to elucidate the mechanism whereby Runx2 and the protein kinase A (PKA) pathway modulate RANKL expression, especially from the viewpoint of their functions in RANKL basic promoter activity and in chromatin structural changes in osteoblastic/stromal cells. Osteoblastic/stromal cell lines derived from normal and Runx2-deficient mice were used to analyze endogenous RANKL gene expression by real-time reverse transcription (RT)-PCR, the acetylation status of the H3 and H4 histone proteins associated with the 5'-flanking region of the RANKL gene by chromatin immunoprecipitation, and the exogenously transfected RANKL gene promoter activity both in the steady-state and under PKA-activated conditions. Here, we demonstrate that Runx2 suppresses steady-state RANKL gene expression by condensing chromatin, while showing a slightly positive effect on RANKL basic promoter activity. Besides acting through the CRE-like region (-0.96 kb) of the RANKL gene promoter, forskolin (FK) treatment transactivates the RANKL gene by antagonizing the function of Runx2, by reducing Runx2 mRNA expression and by opening the chromatin conformation far upstream (more than 40 kb) of the RANKL gene.

RANKL in human periapical granuloma: possible involvement in periapical bone destruction

The cytokine receptor activator of nuclear factor kappaB-ligand (RANKL) has been involved in both the physiological and pathological regulation of osteoclast life span and bone metabolism. Periapical granuloma is a periradicular lesion characterized by periapical bone destruction. The aims of this study were to associate the RANKL mRNA levels to periapical granulomas using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) technique and to determine the specific cell involved in RANKL synthesis. In eight periapical granuloma and eight periodontal ligament samples from periodontally healthy volunteers, RANKL mRNA was detected by real-time RT-PCR. Expression of RANKL on infiltrate leukocytes was further investigated by flow cytometry in six periapical granulomas. Receptor activator of nuclear factor kappaB-ligand mRNA levels were higher in periapical granulomas than in healthy periodontal ligament as its RANKL mRNA cycle threshold (Ct) and DeltaCt were significantly lower than that of controls (33.07 +/- 1.24 vs 36.96 +/- 1.69 and 11.58 +/- 3.02 vs 15.60 +/- 3.31, respectively). A 16.2-fold (2.0-131.6) higher RANKL gene expression was detected in the granulomas compared with the control tissues. We determined by flow cytometry that lymphocytes were the predominant leukocyte cells (53.31%), and monocytes and dendritic cells were the main RANKL synthesizers in granuloma lesions. These data indicate that monocytes synthesized RANKL in periapical granulomas and suggest that RANKL is involved in bone loss associated with periapical lesions.

Response to mechanical strain in an immortalized pre‐osteoblast cell is dependent on ERK1/2

Mechanical strain inhibits osteoclastogenesis by regulating osteoblast functions: We have shown that strain inhibits receptor activator of NF-kappaB ligand (RANKL) expression and increases endothelial nitric oxide synthase (eNOS) and nitric oxide levels through ERK1/2 signaling in primary bone stromal cells. The primary stromal culture system, while contributing greatly to understanding of how the microenvironment regulates bone remodeling is limited in use for biochemical assays and studies of other osteoprogenitor cell responses to mechanical strain: Stromal cells proliferate poorly and lose aspects of the strain response after a relatively short time in culture. In this study, we used the established mouse osteoblast cell line, conditionally immortalized murine calvarial (CIMC-4), harvested from mouse calvariae conditionally immortalized by insertion of the gene coding for a temperature-sensitive mutant of SV40 large T antigen (TAg) and support osteoclastogenesis. Mechanical strain (0.5-2%, 10 cycles per min, equibiaxial) caused magnitude-dependent decreases in RANKL expression to less than 50% those of unstrained cultures. Overnight strains of 2% also increased osterix (OSX) and RUNX2 expression by nearly twofold as measured by RT-PCR. Importantly, the ERK1/2 inhibitor, PD98059, completely abrogated the strain effects bringing RANKL, OSX, and RUNX2 gene expression completely back to control levels. These data indicate that the strain effects on CIMC-4 cells require activation of ERK1/2 pathway. Therefore, the CIMC-4 cell line is a useful alternative in vitro model which effectively recapitulates aspects of the primary stromal cells and adds an extended capacity to study osteoblast control of bone remodeling in a mechanically active environment.

Local RANKL gene transfer to the periodontal tissue accelerates orthodontic tooth movement

It has been reported that not only selective alveolar-bone resorption, but also receptor activator of nuclear factor kappa B ligand (RANKL) expression is induced on the compressed side of an orthodontically moving tooth. Numerous reports have described the pharmacological acceleration of tooth movement (TM) through the activation of osteoclasts. However, because of rapid flush out by blood circulation, daily systemic administration or daily local injection is needed. Previously, we discovered that every-3-days OPG gene transfer to the periodontal-tissue inhibited RANKL-mediated osteoclastogenesis and diminished experimental TM. Therefore, we hypothesized that local RANKL gene transfer into the periodontal tissue would accelerate TM. The upper first molars of 6-week-old male Wistar rats were moved palatally using fixed orthodontic wires. The inactivated hemagglutinating-virus of Japan (HVJ) envelope vector containing the mouse RANKL expression plasmid was injected periodically into the palatal periodontal tissue of the upper first molars during TM. Local RANKL gene transfer significantly enhanced RANKL expression and osteoclastogenesis in periodontal tissue without any systemic effects. The TM rate was significantly increased in the RANKL gene transfer side. In conclusion, we demonstrated that transfer of the RANKL gene to the periodontal-tissue activated osteoclastogenesis and accelerated the amount of experimental TM. Local RANKL gene transfer might be a useful tool not only for shortening orthodontic treatment, but also for moving ankylosed teeth where teeth, fuse to the surrounding bone.

Expression profile of genes related to osteoclastogenesis in mouse growth plate and articular cartilage

Based on developmental fate and function, cartilage tissue is broadly classified into transient cartilage (e.g. growth plate, GP) and permanent cartilage (e.g. articular cartilage, AC). The former eventually disappears and is replaced by bone during the endochondral ossification process, whereas the latter retains its permanency. Osteo(chondro)clasts, multinucleated giant cells of the monocyte/macrophage lineage, are selectively induced in the GP during endochondral ossification and play central roles in the resorption of cartilagenous matrices. The aim of this study was to investigate the factors determining the GP-specific recruitment of osteo(chondro)clasts. We especially focused on the expression pattern of the receptor activator of NF-kappaB ligand (RANKL), an essential factor for osteo(chondro)clast differentiation, and on that of epigenetic and transcriptional factors affecting RANKL gene expression. Knee joints of male BALB/c mice aged 8 weeks were dissected and subjected to immunohistochemical analysis using anti-RANKL, Runx2, Dlx5 and Msx2 antibodies. The methylation status of the mouse RANKL gene promoter in both the GP and the AC was analyzed by sodium bisulfite mapping using microdissected mouse tissue. The expression of BMP-2, -3, -4, -6 and type X collagen mRNA was examined by in situ hybridization (ISH). At the boundary between the calcifying cartilage and the hypertrophic chondrocytes of the GP, RANKL-expressing chondrocytes overlapped those expressing Runx2, Dlx5 and Msx2, near numerous osteo(chondro)clasts. Although similar BMP-2 and -4 expression was observed in chondrocytes in both the GP and the AC as well as in maturing osteoblasts, a rather restricted BMP-6 expression pattern was observed in resting and proliferating chondrocytes in the GP. On the other hand, sodium bisulfite mapping showed that mostly non-CpG methylation was similarly scattered in a non-specific manner in chondrocytes in the GP and the AC. Taken together with the fact that putative Runx2 binding elements are located in the RANKL promoter, our data suggest that Runx2, an essential transcription factor for skeletal development, is also a key regulator of RANKL expression in chondrocytes in the GP. Furthermore, a selective and sequential expression of a subset of BMP and of transcription factors may define the expression pattern of RANKL through Runx2.

Chronology and regulation of gene expression of RANKL in the rat dental follicle

Tooth eruption in the rat requires bone resorption resulting from a major burst of osteoclastogenesis on postnatal day 3 and a minor burst of osteoclastogenesis on postnatal day 10 in the alveolar bone of the first mandibular molar. The dental follicle regulates the major burst on postnatal day 3 by down-regulating its osteoprotegerin (OPG) gene expression to enable osteoclastogenesis to occur. To determine the role of receptor activator of nuclear factor-kappa B ligand (RANKL) in tooth eruption, its gene expression was measured on postnatal days 1-11 in the dental follicle. The results show that RANKL expression was significantly elevated on postnatal days 9-11 in comparison to low expression levels at earlier time-points. As OPG expression is high at this latter time-point, this increase in RANKL expression would be needed for stimulating the minor burst of osteoclastogenesis. Tumor necrosis factor-alpha enhances RANKL gene expression in vitro and it may be responsible for up-regulating RANKL in vivo. Transforming growth factor-beta1 and interleukin-1alpha also enhance RANKL gene expression in vitro but probably have no effect in vivo because they are maximally expressed early. Bone morphogenetic protein-2 acts to down-regulate RANKL expression in vitro and, in vivo, may promote alveolar bone growth in the basal region of the tooth.

Areca nut extracts modulated expression of alkaline phosphatase and receptor activator of nuclear factor κB ligand in osteoblasts

Areca chewers have a higher prevalence of periodontal diseases than non-chewers. This study was to determine the possible effects of ripe areca nut extracts (rANE) on viability and gene expression of alkaline phosphatase (ALP), receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in human osteoblasts. The effects of rANE on cell viability of osteoblast-like MG63 cells were determined using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) that measures metabolic activity. Gene expression of ALP, RANKL and OPG was examined using reverse transcription-polymerase chain reaction. ALP activity and RANKL protein were further examined using substrate assay and confocal laser scanning microscopy, respectively. Relative viability was reduced to approximately 50% when 25 microg/ml of rANE was used. The expression of OPG mRNA in MG63 cells was not altered by rANE. However, decreased levels of mRNA and enzyme activity of ALP were observed. Moreover, the expressions of mRNA and protein of RANKL were stimulated by rANE in a dose-dependent manner. The rANE affected morphology and viability of osteoblasts. We also present novel evidence demonstrating that areca nut may compromise the periodontal health of areca chewers by suppression of ALP gene expression and elevation of RANKL gene expression in osteoblasts.

Regulation of RANKL promoter activity is associated with histone remodeling in murine bone stromal cells.

Receptor activator of NFkappa-B ligand (RANKL) is essential for osteoclast formation, function, and survival. Although RANKL mRNA and protein levels are modulated by 1,25(OH)2D3 and other osteoactive factors, regulatory mechanisms remain unclear. In this study, we show that 2 kb or 2 kb plus exon 1 of a RANKL promoter sequence conferred neither 1,25(OH)2D3 response nor tissue specificity. The histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (SB), however, strongly increased RANKL promoter activity. A series of 5'-deleted RANKL promoter constructs from 2,020 to 110 bp showed fourfold increased activity after TSA treatment. TSA also dose dependently enhanced endogenous RANKL mRNA expression with 50 microM of TSA treatment causing equivalent RANKL expression to that seen with 1 nM 1,25(OH)2D3. Using a chromatin immunoprecipitation (ChIP) assay we showed that TSA significantly enhanced association of both acetylated histone H3 and H4 on the RANKL promoter, with H4 > H3. A similar increase in acetylated histone H4 on the RANKL gene locus was seen after 1,25(OH)2D3 treatment, but ChIP assay did not reveal localization of VDR/RXR heterodimers on the putative VDRE of the RANKL promoter. To explore the role of H4 acetylation of 1,25(OH)2D3 stimulated RANKL, we added both TSA and 1,25(OH)2D3 together. While the combination further increased acetylation of H4 on the RANKL locus, surprisingly, TSA inhibited 1,25(OH)2D3-induced RANKL mRNA expression by 70% at all doses of 1 ,25(OH)2D3 studied. These results suggest that TSA increases of endogenous expression of RANKL involve enhanced acetylation of histones on the proximal RANKL promoter. Preventing deacetylation, however, blocks 1,25(OH)2D3 action on this gene. Chromatin remodeling is therefore involved in RANKL expression.

Functional role for heat shock factors in the transcriptional regulation of human RANK ligand gene expression in stromal/osteoblast cells.

RANK Ligand (RANKL) is a critical osteoclastogenic factor that is expressed on stromal cells and osteoblasts. Most resorption stimuli induce osteoclast formation by modulating RANKL gene expression in marrow stromal/osteoblast cells. However, it is unclear how these stimuli modulate RANKL gene expression in the bone microenvironment. To characterize the transcriptional control of human RANKL gene expression in stromal/osteoblast cells, we PCR-amplified and cloned a 2-kb 5'-flanking sequence of the RANKL gene, using normal human osteoblast derived genomic DNA as a template. Sequence analysis identified the presence of several potential Heat Shock Factor (HSF) responsive elements (HSE) in the human RANKL gene promoter region. Co-expression of HSF-1 or HSF-2 with the RANKL gene promoter-luciferase reporter plasmid in human osteoblastic cells (NOBC) demonstrated a 2-fold and 4.5-fold increase in promoter activity, respectively. RT-PCR analysis for HSF-1 and 2 mRNA expression in human bone marrow-derived stromal cells (SAKA-T) and osteoblast cells detected only HSF-2 expression. As evident from EMSA analysis, in contrast to 1,25(OH)(2)D(3) SAKA-T cells treated with b-FGF demonstrated increased levels of HSF-2 binding to the HSE present in the RANKL gene promoter region. Immunocytochemical staining further confirmed nuclear localization of HSF-2 in both SAKA-T transformed stromal cells and human bone marrow derived primary stromal/preosteoblastic cells in response to b-FGF treatment. Furthermore, b-FGF treatment of SAKA-T cells transfected with the luciferase reporter plasmid containing the hRANKL HSE region (-2 kb to -1275 bp) upstream to a heterologous promoter showed increased levels of transactivation. Western blot analysis further demonstrated enhanced levels of RANKL expression and HSP-27 phosphorylation in SAKA-T cells treated with b-FGF. In addition, overexpression of HSF-2 in SAKA-T cells resulted in a 5-fold increase in the levels of RANKL expression in these cells. These data further suggest that HSF-2 is a downstream target of b-FGF to induce RANKL expression in stromal/osteoblast cells, and that HSF may play an important role in modulating RANKL gene expression in the bone microenvironment.

Vitamin D3 supports osteoclastogenesis via functional vitamin D response element of human RANKL gene promoter.

Receptor activator of NF-kappaB ligand (RANKL) has been identified as requisite for osteoclastogenesis. To elucidate the molecular mechanism that conducts its catabolic action on bone, the effect of 1alpha,25 dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) on osteoclastogenesis and RANKL mRNA expression was examined by coculture, RT-PCR and nuclear run-on studies. By accelerating the transcription rate of the RANKL gene in SaOS2 osteoblastic cells, 1alpha,25(OH)(2)D(3) enhanced in vitro osteoclast formation from peripheral monocytes. Cloning and characterization of the 5'-flanking region of the human RANKL gene revealed that the basic promoter comprises inverted TATA- and CAAT-boxes flanked by RUNX2 binding sites. Both electrophoresis mobility shift assay (EMSA) and transfection studies demonstrated that 1alpha,25(OH)(2)D(3) activated human RANKL promoter through vitamin D responsive elements (VDRE) located at -1584/-1570 by binding VDR and RXRalpha heterodimers in a ligand-dependent manner. The results provide direct evidence that 1alpha,25(OH)(2)D(3) augments osteoclastogenesis by transactivating the human RANKL gene in osteoblastic cells through VDRE.

Expression of RANKL and OPG mRNA in periodontal disease: possible involvement in bone destruction.

Periodontitis is a complex, multifactorial process affected by bacterial plaque-components and host defense mechanisms. Inflammation of the periodontitium may lead the destruction of the underlying ligament and alveolar bone. Receptor activator of NF-kappaB ligand (RANKL), a novel TNF receptor-related protein is an important factor for osteoclast differentiation and activation. Given osteolysis by osteoclast has been demonstrated in periodontitis, we hypothesized that RANKL expression may be associated with bone destruction in periodontitis. We used semi-quantitative RT-PCR to compare the gene expression of RANKL and osteoprogerin (OPG), a decoy receptor of RANKL, between moderate and advanced periodontitis, and healthy subjects. The level of RANKL mRNA was highest in advanced periodontitis. In contrast, the level of OPG mRNA in both advanced and moderate periodontitis was lower than that in the healthy group. It appears that the ratio of RANKL to OPG mRNA in periodontitis has increased. To determine the localization of RANKL gene transcripts in gingival tissue at the cellular level, in situ hybridization was performed using digoxigenin-labeled specific riboprobes. RANKL mRNA was expressed in inflammatory cells, mainly lymphocyte and macrophages. In addition, proliferating epithelium in the vicinity of inflammatory cells expressed high levels of RANKL mRNA. In short, our data suggest that up regulation of RANKL mRNA in both inflammatory cells and epithelium may be associated with the activation of osteoclastic bone destruction in periodontitis.