Receptor Activator Of NF-kappaB Ligand Gene Research Articles

Association of RANKL and EGFR gene expression with bone metastases in patients with metastatic non-small cell lung cancer.

Bone metastases are frequent in patients with non-small cell lung cancer (NSCLC). The receptor activator of Nuclear Factor κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) pathway is important in bone metastases development. Furthermore, epidermal growth factor receptor (EGFR) signaling promotes osteoclast formation and stimulation. The understanding of the biological mechanism of bone metastases development might have implications for treatment strategies. Therefore, we studied whether there is an association between EGFR, RANKL, RANK and OPG gene expression in the tumor and presence of bone metastases in patients with NSCLC. From an updated multicenter study, including patients with EGFR mutated (EGFR+), Kirsten rat sarcoma (KRAS+) and EGFR/KRAS wildtype metastatic NSCLC, all patients with available formalin-fixed paraffin-embedded (FFPE) tumor samples were selected. Ribonucleic Acid (RNA) was isolated from these samples and gene expressions of EGFR, RANKL, OPG and RANKL were determined via quantitative Polymerase Chain Reaction (qPCR). Data on demographics, histology and molecular subtyping, sample origin, presence of bone metastasis, SREs and bone progression were collected. Primary endpoint was relation between EGFR, RANK, RANKL, OPG gene expression, RANKL: OPG ratio and bone metastases. In 73/335 (32% EGFR+, 49% KRAS+, 19% EGFR/KRAS wildtype) samples from unique patients, gene expression analysis could be performed. Of these 73 patients, 46 (63%) had bone metastases at diagnosis or developed bone metastases during the disease course. No association was found between EGFR expression and presence of bone metastases. Patients with bone metastases had a significantly higher RANKL expression and RANKL: OPG ratio compared to those without. An increased RANKL: OPG ratio resulted in a 1.65x increased risk to develop bone metastases, especially in the first 450 days after diagnosis of metastatic NSCLC. Increased RANKL gene expression and RANKL: OPG ratio, but not EGFR expression, was associated with presence of bone metastases. Additionally, an increased RANKL: OPG gene ratio was associated with a higher incidence of bone metastases development.

Targeting ROS-induced osteoblast senescence and RANKL production by Prussian blue nanozyme based gene editing platform to reverse osteoporosis

Blocking the reactive oxygen species (ROS) induced osteoblast senescence and massive RANKL release is the key to alleviate the vicious osteoporotic micro-environment in aged skeleton. In this study, we constructed a composite nanoparticle by confining the growth of Prussian blue nanozyme (PBzyme) on the surface of a sub-50 nm hollow mesoporous silica nanoparticles (HPB), which presented strong ROS scavenging ability. The abundant mesoporous structure made it available to combine receptor activator of nuclear factor kappa-B ligand (RANKL) CRISPR/Cas9 plasmid and skeletal targeting agent alendronate (HPB@RC-ALN) together to modulate the osteoporotic micro-environment. Our results indicate HPB@RC-ALN confined ROS generation and alleviated osteoblast senescence as revealed by dihydroethidium and senescence-associated β-galactosidase staining. The rejuvenated osteoblasts presented robust osteogenic ability as shown by alkaline phosphatase and alizarin red staining in vitro, as well as the micro-computed tomography (μCT) in vivo. Meanwhile, efficient transfection of CRISPR/Cas9 plasmid by HPB@RC-ALN precisely achieved RANKL gene editing target and knocked the RANKL gene out in osteoblasts as confirmed by T7E1 digestion and western blot assays. Hence, the osteoclast formation was significantly suppressed as shutting down the RANKL production by HPB@RC-ALN in the senescent osteoblast. At last, the μCT revealed fully reversed bone volume in ovariectomized mice after HPB@RC-ALN injection into the bone remodeling site. Overall, the constructed nanozyme based gene editing platform achieved bidirectional regulation of osteoblast-osteoclast through rescuing cellular senescence and blocking RANKL production. This strategy provides a new theoretical base for osteoporosis management.

Effects of Alendronate and Dexamethasone on Osteoclast Gene Expression and Bone Resorption in Mouse Marrow Cultures.

Osteoclasts are cells whose main function is the resorption of bone matrix. However, several factors, including medications, can interfere with the resorption process. Alendronate (ALN), a nitrogen-containing type of bisphosphonate, and dexamethasone (DEX), a glucocorticoid, are drugs that may affect the resorption activity. The aim of this study is to investigate the effects of ALN, and/or DEX on osteoclast gene expression and resorption activity in primary mouse marrow cultures stimulated with 1,25-dihydroxyvitamin D3, a model for the bone microenvironment. Cultures were treated only with ALN (10-5 M), DEX (10-6 M), and with a combination of both agents. Viability assays performed at days 5, 7, and 9 showed the highest number of viable cells at day 7. All the following assays were then performed at day 7 of cell culture: tartrate resistant acid phosphatase (TRAP) histochemistry, receptor activator of nuclear factor kappa B ligand (RANKL) immunofluorescence, osteoprotegerin (OPG), and RANKL gene expression by qPCR and resorption analysis by scanning electron microscopy. Treatment with ALN, DEX, and the combination of both did not promote significant changes in the number of TRAP+ cells, although larger giant cells were detected in groups treated with DEX. DEX treatment increased the gene expression of RANKL and reduced OPG. The treatment with ALN reduced the depth of the resorption pits, but their inhibitory effect was less effective when administered with DEX.

Effects of Er:YAG laser irradiation of different titanium surfaces on osteoblast response

The aim of this in vitro study was to evaluate the effects of erbium-doped yttrium aluminum garnet (Er:YAG) laser irradiation on titanium surface topography and the proliferation and differentiation of osteoblasts using standard clinical treatment settings. Er:YAG laser irradiation at two levels ((1): 160 mJ, pulse at 20 Hz; (2): 80 mJ, pulse at 20 Hz) was applied to moderately rough and smooth titanium disks before MG-63 osteoblast-like cells were cultured on these surfaces. Titanium surface and cell morphology were observed by scanning electron microscopy. Cell proliferation/viability was measured by CCK-8 test. Gene expression of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B ligand (RANKL), and collagen type 1 was measured by qPCR, and OPG and OC protein production was determined by enzyme-linked immunosorbent assay. Treatment with Er:YAG laser at 160 mJ/20 Hz markedly caused heat-induced fusion of titanium and cell condensation on moderately rough surfaces, but not in smooth surfaces. MG-63 proliferation/viability decreased after 5 days in moderately rough surfaces. The expression of ALP, OC, OPG, and collagen type 1 was unaffected by laser treatment at 160 mJ/20. Laser irradiation at 80 mJ/20 Hz enhanced RANKL gene expression after 5 days in moderately rough surfaces. Study results suggest that Er:YAG laser irradiation at clinically relevant setting has no essential effect on osteogenic gene and protein expression of osteoblasts. However, surface structure, cell attachment, and proliferation are influenced by both treatment protocols, which implies that caution should be taken in the clinical treatment of peri-implant diseases when Er:YAG laser is used.

Structure of a DNA G-Quadruplex Related to Osteoporosis with a G-A Bulge Forming a Pseudo-loop.

Bone remodeling is a fine-tuned process principally regulated by a cascade triggered by interaction of receptor activator of NF-κB (RANK) and RANK ligand (RANKL). Excessive activity of the RANKL gene leads to increased bone resorption and can influence the incidence of osteoporosis. Although much has been learned about the intracellular signals activated by RANKL/RANK complex, significantly less is known about the molecular mechanisms of regulation of RANKL expression. Here, we report on the structure of an unprecedented DNA G-quadruplex, well-known secondary structure-mediated gene expression regulator, formed by a G-rich sequence found in the regulatory region of a RANKL gene. Solution-state NMR structural study reveals the formation of a three-layered parallel-type G-quadruplex characterized by an unique features, including a G-A bulge. Although a guanine within a G-tract occupies syn glycosidic conformation, bulge-forming residues arrange in a pseudo-loop conformation to facilitate partial 5/6-ring stacking, typical of G-quadruplex structures with parallel G-tracts orientation. Such distinctive structural features protruding from the core of the structure can represent a novel platform for design of highly specific ligands with anti-osteoporotic function. Additionally, our study suggests that the expression of RANKL gene may be regulated by putative folding of its G-rich region into non-B-DNA structure(s).

Toll-like receptor 7 regulates osteoclastogenesis in rheumatoid arthritis.

This study aimed to determine the regulatory role of toll-like receptor 7 (TLR7) in receptor activator of nuclear factor kappa-B ligand (RANKL) production and osteoclast differentiation in rheumatoid arthritis (RA). In confocal microscopy, the co-expression of TLR7, CD55 and RANKL was determined in RA synovial fibroblasts. After RA synovial fibroblasts were treated with imiquimod, the RANKL gene expression and protein production were determined by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Osteoclastogenesis from peripheral blood CD14+ monocytes which were cultured with imiquimod was assessed by determining the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. The signal pathways mediating the TLR7-induced RANKL expression and osteoclastogenesis were analysed after inhibition of intracellular signal molecules and their phosphorylation. Imiquimod stimulated the expression of TLR7 and RANKL and production of RANKL in RA synovial fibroblasts, increasing the phosphorylation of TRAF6, IRF7, mitogen-activated protein kinases (MAPK), c-Jun and NFATc1. When CD14+ monocytes were cultured with imiquimod or co-cultured with imiquimod-pre-treated RA synovial fibroblasts, they were differentiated into TRAP+ multinucleated osteoclasts in the absence of RANKL. TLR7 activation-induced osteoclastogenesis in RA through direct induction of osteoclast differentiation from its precursors and up-regulation of RANKL production in RA synovial fibroblasts. Thus, the blockage of TLR7 pathway could be a promising therapeutic strategy for preventing bone destruction in RA.

-643C > T RANKL gene polymorphism is associated with osteoporosis in Tunisian postmenopausal women

Objectives: The dynamic nature of the skeleton is achieved by a remodeling process. Receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) stimulates bone resorption by activating RANK signaling. Therefore it is considered as a candidate gene regulating susceptibility to osteoporosis. In the current study, we have investigated the association between the RANKL gene -693G > C and -643 C > T polymorphisms and bone mineral density (BMD) in a population of postmenopausal Tunisian women.Methods: Polymorphic sites in RANKL gene (rs9533155 -693G > C and rs9533156 -643 C > T polymorphisms) were determined using PCR-RFLP analysis in 566 postmenopausal Tunisian women. All statistical analysis were examined by SPSS software.Results: We have detected a significant difference in lumbar spine and hip BMD for -643C > T genotypes. For -693G > C genotypes, a significant difference was detected only in hip BMD. The distribution of -643C > T genotypes and alleles between three groups (osteoporotic, osteopenic and normal women) revealed a significant association of the TT genotype with development of osteoporosis (p = 0.01; odds ratio 2.15), although for the -693G > C polymorphism, no significant results were found.Conclusion: We have demonstrated the association of the -643C > T polymorphism with BMD variation and osteoporosis risk in postmenopausal Tunisian women.

OPG and RANKL polymorphisms are associated with alcohol-induced osteonecrosis of the femoral head in the north area of China population in men.

Alcohol-induced osteonecrosis of the femoral head (ONFH) is an important pathogenesis of nontraumatic ONFH. However, the mechanisms of the pathogenesis are still unknown. Osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B ligand (RANKL) have been implicated in multiple functions including blocking osteoclast maturation, controlling vascular calcifications, and promoting tumor growth and metastasis. The purpose of this article was to explore the association between OPG and RANKL gene variants and alcohol-induced ONFH. Six hundred seventy male subjects (335 patients and 335 normal individuals) were enrolled in our study. We selected 24 single-nucleotide polymorphisms (SNPs) to evaluate the association between genetic susceptibility variants and alcohol-induced ONFH using the chi-square test and gene model analysis. Overall, the OPG SNPs (rs1032128 and rs11573828) were associated with the strongest increased risk of alcohol-induced ONFH in the recessive model (rs1032128: odds ratio [OR] 1.49, 95% confidence interval [CI] 1.00–2.22, P = 0.04 for G/A; rs11573828: OR 3.32, 95% CI 1.07–10.30, P = 0.03 for T/C). The RANKL SNP rs2200287 was also an increased risk factor (OR 3.65, 95% CI 1.53–8.47, P = 0.003 for T/C) in the recessive model. The rs11573856, rs3134056, and rs1564861 SNPs were considered protective factors for alcohol-induced ONFH. We concluded that OPG and RANKL polymorphisms were associated with the occurrence of alcohol-induced ONFH.

Effect of compressive loading and incubation with clodronate on the RANKL/OPG system of human osteoblasts.

In vivo studies have shown that bisphosphonates result in slow rates of orthodontic tooth movement. This study investigated whether clodronate modifies the impact of mechanical loading on the RANKL/OPG system of human osteoblasts. Osteoblasts were cultured in vitro with 0.5 or 5.0 µM clodronate for 48 h and/or subjected to 3 h of compressive loading at 34.9 g/cm(2). Cell viability was determined by MTT assay. Real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and immunocytochemical staining were used to analyze the cells for their production of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) at the transcriptional and protein levels. Compressive loading did not affect osteoblast viability in a significant way. Clodronate (5.0 µM) mildly reduced the viability of both compressed and uncompressed cells. Compressive loading induced a 4.2-fold increase in RANKL gene expression, while clodronate led to a concentration-dependent inhibition of this effect (1.8-fold increase at 5.0 µM). OPG gene expression was decreased by compressive loading both in the presence of 0.5 µM clodronate and in the absence of clodronate, and OPG protein synthesis in the compressed cells was significantly decreased in the presence of clodronate. Immunocytochemical staining revealed an increase of RANKL protein synthesis in compressed cells, while clodronate and cell compression reduced this increase. This study demonstrates that clodronate decreases the compression-induced RANKL/OPG ratio expressed by human osteoblasts. Reported in vivo findings of reduced osteoclast numbers on the compression side of orthodontic tooth movement under the action of clodronate-and the associated slow rate of tooth movement-might be attributable not only to a direct impact on osteoclasts but also to changes in osteoblast-osteoclast interaction resulting from the presence of clodronate.

Association of receptor activator of nuclear factor-kappaB ligand (RANKL) gene polymorphisms with the susceptibility to ankylosing spondylitis: a case-control study

ObjectivesTo investigate the association between receptor activator of nuclear factor-kappaB ligand (RANKL) gene polymorphisms and the susceptibility to ankylosing spondylitis (AS) in a Chinese Han population. MethodsThree hundred and fifty-two AS patients and 299 age- and gender-matched controls were recruited in this study. Peripheral blood samples were collected from all the subjects and the genomic DNA was then extracted. Three single nucleotide polymorphisms (SNPs) of the RANKL gene (rs2277438, rs7984870 and rs9533156) were genotyped using the TaqMan assay. The frequencies of alleles and genotypes were compared between AS patients and normal controls. ResultsThe distributions of genotype frequencies in rs2277438 were significantly different between AS patients and normal controls (P < 0.05). The frequency of G allele of SNP rs2277438 in AS patients was significantly higher than that in normal controls (P < 0.05). The frequencies of genotypes with G allele (GG and AG) were significantly higher in AS patients when compared with normal controls (OR = 1.573, 95 % CI 1.151-2.150, P < 0.05). Neither the genotype frequencies nor the allele frequencies of rs7984870 and rs9533156 were found to be significantly different between AS patients and normal controls (P > 0.05). ConclusionsThe current study demonstrated that SNP rs2277438 of the RANKL gene was associated with the susceptibility of AS in a Chinese Han population. Genotypes with G allele (GG and AG) were identified as the risk factors for the occurrence of AS.

Labisia pumila regulates bone-related genes expressions in postmenopausal osteoporosis model.

BackgroundLabisia Pumila var. alata (LPva) has shown potential as an alternative to estrogen replacement therapy (ERT) in prevention of estrogen-deficient osteoporosis. In earlier studies using postmenopausal model, LPva was able to reverse the ovariectomy-induced changes in biochemical markers, bone calcium, bone histomorphometric parameters and biomechanical strength. The mechanism behind these protective effects is unclear but LPva may have regulated factors that regulate bone remodeling. The aim of this study is to determine the bone-protective mechanism of LPva by measuring the expressions of several factors involved in bone formative and resorptive activities namely Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor kappa-B Ligand (RANKL), Macrophage-Colony Stimulating Factor (MCSF) and Bone Morphogenetic Protein-2 (BMP-2).MethodsThirty-two female Wistar rats were randomly divided into four groups: Sham-operated (Sham), ovariectomized control (OVXC), ovariectomized with Labisia pumila var. alata (LPva) and ovariectomized with ERT (Premarin®) (ERT). The LPva and ERT were administered via daily oral gavages at doses of 17.5 mg/kg and 64.5 μg/kg, respectively. Following two months of treatment, the rats were euthanized and the gene expressions of BMP-2, OPG, RANKL and MCSF in the femoral bones were measured using a branch - DNA technique.ResultsThe RANKL gene expression was increased while the OPG and BMP-2 gene expressions were reduced in the OVXC group compared to the SHAM group. There were no significant changes in the MCSF gene expressions among the groups. Treatment with either LPva or ERT was able to prevent these ovariectomy-induced changes in the gene expressions in ovariectomized rats with similar efficacy.ConclusionLPva may protect bone against estrogen deficiency-induced changes by regulating the RANKL, OPG and BMP-2 gene expressions.

SOCS-1/3 participation in FGF-2 signaling to modulate RANK ligand expression in paget's disease of bone

Paget's disease of bone (PDB) is a chronic focal skeletal disorder characterized by excessive bone resorption followed by disorganized new bone formation. Measles virus nucleocapsid (MVNP) is implicated in pathogenesis of PDB. RANK ligand (RANKL), a critical osteoclastogenic factor expressed on bone marrow stromal/preosteoblast cells is upregulated in PDB. We recently demonstrated that fibroblast growth factor-2 (FGF-2) which induces RANKL expression is elevated in PDB. In this study, we hypothesized that FGF-2 modulates suppressors of cytokine signaling (SOCS) to induce RANKL expression in PDB. We identified increased levels of SOCS-1/3 mRNA expression in bone marrow mononuclear cells derived from patients with PDB compared to normal subjects. Interestingly, conditioned media obtained from MVNP transduced osteoclast progenitor cells significantly increased SOCS-1/3 mRNA expression in stromal/preosteoblast cells. We next examined if SOCS participates in FGF-2 signaling to modulate RANKL gene expression. We showed that FGF-2 stimulation significantly increased SOCS-1/3 expression in human bone marrow stromal/preosteoblast cells. In addition, co-expression of SOCS-1/3 with hRANKL gene promoter-luciferase reporter plasmid in marrow stromal cells demonstrated a significant increase in promoter activity without FGF-2 stimulation. Furthermore, siRNA inhibition of STAT-1 suppresses FGF-2 increased SOCS-1/3 expression in these cells. Thus, our results suggest that SOCS participates in FGF-2 modulation of RANKL expression in PDB.

Mechanical loading prevents the stimulating effect of IL-1β on osteocyte-modulated osteoclastogenesis

Inflammatory diseases such as rheumatoid arthritis are often accompanied by higher plasma and synovial fluid levels of interleukin-1β (IL-1β), and by increased bone resorption. Since osteocytes are known to regulate bone resorption in response to changes in mechanical stimuli, we investigated whether IL-1β affects osteocyte-modulated osteoclastogenesis in the presence or absence of mechanical loading of osteocytes.MLO-Y4 osteocytes were pre-incubated with IL-1β (0.1–1ng/ml) for 24h. Cells were either or not subjected to mechanical loading by 1h pulsating fluid flow (PFF; 0.7±0.3Pa, 5Hz) in the presence of IL-1β (0.1–1ng/ml). Conditioned medium was collected after 1h PFF or static cultures. Subsequently mouse bone marrow cells were seeded on top of the IL-1β-treated osteocytes to determine osteoclastogenesis. Conditioned medium from mechanically loaded or static IL-1β-treated osteocytes was added to co-cultures of untreated osteocytes and mouse bone marrow cells. Gene expression of cysteine-rich protein 61 (CYR61/CCN1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) by osteocytes was determined immediately after PFF.Incubation of osteocytes with IL-1β, as well as conditioned medium from static IL-1β-treated osteocytes increased the formation of osteoclasts. However, conditioned medium from mechanically loaded IL-1β-treated osteocytes prevented osteoclast formation. Incubation with IL-1β upregulated RANKL and downregulated OPG gene expression by static osteocytes. PFF upregulated CYR61, RANKL, and OPG gene expression by osteocytes.Our results suggest that IL-1β increases osteocyte-modulated osteoclastogenesis, and that mechanical loading of osteocytes may abolish IL-1β-induced osteoclastogenesis.

Residents' journal review

Residents' journal review

Genetic Polymorphisms of the RANKL and OPG Genes Associated with Myeloma

Abstract 2911The bone disease in myeloma is the result of the complex interactions between myeloma cells and the bone marrow osteoclasts plus other accessory cells and microenvironmental components. The receptor activator of NfkappaB (RANK) and osteoprotegerin (OPG) cascade system have been reported to be an essential pathway in the osteoclastogenesis. Genetic variations in the genes coding for RANK, RANK ligand (RANKL), and OPG are supposed to play a role in the susceptibility of myeloma.In the present case control study genomic DNA was obtained in 217 myeloma patients including smoldering myeloma (age: mean 62, 33–88) and 516 healthy controls (healthy blood donors, HC; age mean: 44, 18–65). We studied six single-nucleotide-polymorphisms (SNPs) in the genes of RANK (2 SNPs, rs1805034, rs35211496), OPG (2 SNPs, rs3102735, rs2073618), and of RANKL (2 SNPs, rs9533156, rs2277438) using TaqMan assay guided polymerase chain reaction for the respective SNPs. The genotype and allelic frequencies comparing myeloma patients with HC were analyzed with χ2 test for 2×3 and 2×2 tables, respectively.The genotype distributions of the SNP rs2277438 in the RANKL gene and SNP rs3102735 in the OPG gene differed highly significantly (p=0.0001 and p=0.005, respectively) between myeloma patients and HC (table 1). No significantly increased risk was detected in the other SNP analyzed in the study. A subgroup analysis of the myeloma patients stratified into beta2 microglobulin levels lower or higher than 3.5 mg/l showed lower values to be significantly associated with the minor allele of the SNP rs3102735 in the OPG gene (major vs. minor allele in myeloma vs. HC: 77/23% vs. 89/11%; OR 2.49; CI 1.38–4.49). Further stratification into gender, serum calcium levels, hemoglobin values at diagnosis showed no differences.Table 1Myeloma patientsHealthy controls (HC)OR (CI)pRANKL rs2277438Allelen = 434 (%)n = 1024 (%)1324 (75)860 (84)1.7790.00012110 (25)164 (16)(1.373–2.341)Genotypen = 217 (%)n = 512 (%)11132 (61)360 (70)0.00011260 (28)140 (27)2225 (12)12 (2)OPG rs 3102735Allelen = 420 (%)n = 1028 (%)167 (16)109 (11)1.6000.0052353 (84)919 (89)(1.153–2.222)Genotypen = 210 (%)n = 514 (%)119 (4)5 (1)0.0051249 (23)99 (19)22152 (72)410 (80)RANK = receptor activator of NfkappaB, RANKL = receptor activator of NfkappaB ligand, OPG = osteoprotegerin, OR = odds ratio, CI = confidence intervals 95%To the best of our knowledge, this is the first study reporting a highly significant association of the SNP rs2277438 of the RANKL gene and the SNP rs3102735 in the OPG gene, respectively, with the susceptibility to develop myeloma in the Caucasian population. Both SNPs are associated with an increased risk for myeloma. However, myeloma patients carrying the minor allele of OPG SNP rs3102735 developed lower levels of the proliferation marker beta2 microglobulin. The impact of the OPG SNP rs3102735 (location: promoter region, chromosome 8q24) or of the SNP rs2277438 located in intron 1 (chromosome 13q14.11), a potentially regulatory region of the RANKL gene, on the regulation of RANKL expression is unclear. Disclosures:No relevant conflicts of interest to declare.

Activation of cannabinoid receptor CB2 regulates osteogenic and osteoclastogenic gene expression in human periodontal ligament cells

Cannabinoid receptor CB2, expressed in osteoblasts and osteoclasts, plays a crucial role in the regulation of bone metabolism. Since periodontal ligament (PDL) cells can differentiate into osteoblasts, this study was undertaken to investigate CB2 expression and the effect of CB2 activation on osteogenic differentiation of PDL cells. Human PDL (hPDL) cells were obtained from extracted teeth of periodontally healthy subjects. Expression of CB2 was observed in hPDL cells by RT-PCR, Western blotting and immunofluorescence assay. Then hPDL cells were treated with a CB2-specific agonist, HU-308 (10(-7) m), for 12, 24, 48 or 72 h. The mRNA expressions of osteogenic genes, such as runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteopontin (OPN), alkaline phosphatase (ALP), osteocalcin (OC) and collagen type I (COL I), and osteoclastogenic genes, including osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL), were examined using quantitative real-time PCR analysis. A mineralization assay was performed in hPDL cells in mineralization conditions with or without HU-308. Expression of CB2 mRNA and protein was detected in hPDL cells. HU-308 enhanced the mRNA levels of the above osteogenic genes. Expression of the OPG gene was up-regulated, whereas RANKL gene expression was down-regulated, contributing to the elevated OPG/RANKL ratio. Accelerated mineralization was observed in hPDL cells in mineralization conditions with HU-308. Our findings demonstrate that activation of CB2 is able to enhance osteogenic differentiation of hPDL cells and potentially create a favorable osteogenic microenvironment. This implies that CB2 might play an important role in alveolar bone metabolism.

Genetic Variations in Genes Encoding RANK, RANKL, and OPG in Rheumatoid Arthritis: A Case-Control Study

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the joints, which may lead to structural damage of the cartilage and bone. The receptor activator of nuclear factor-kappaB (RANK) and the osteoprotegerin (OPG) cascade system have been reported to be essential in osteoclastogenesis. Genetic variations in the genes coding for RANK, RANK ligand (RANKL), and OPG are thought to play roles in the susceptibility to RA. In our case-control study, genomic DNA was obtained from 534 patients with RA who fulfilled the American College of Rheumatology 1987 criteria and 516 healthy control blood donors (HC). We studied 7 single-nucleotide polymorphisms (SNP) in the genes of RANK (2 SNP: rs1805034, rs35211496), OPG (2 SNP: rs3102735, rs2073618), and RANKL (3 SNP: rs9533156, rs2277438, rs1054016) using TaqMan assay-guided polymerase chain reaction. Genotype and allelic frequencies comparing RA patients with HC were analyzed by chi-square test for 2 x 3 and 2 x 2 tables, respectively. Genotype distributions of the SNP rs35211496 in the RANK gene as well as the SNP rs2277438 in the RANKL gene differed significantly between patients with RA and HC. The frequency of the minor allele of rs9533156 of RANKL was significantly higher in patients with RA than in HC (OR 0.84, 95% CI 0.71-0.99, p = 0.047). Multivariate analysis adjusted to sex and investigating SNP demonstrated a significantly elevated risk for RA associated with the major allele in the RANK SNP rs35211496 (p = 0.0231) and with the minor allele in the RANKL SNP rs2277438 (p = 0.0092). No significantly increased risk was detected in the other SNP. The minor allele of the RANK SNP rs35211496 may be protective against RA, while the minor alleles of the RANKL SNP rs2277438 may increase susceptibility to RA.

The effect of the alendronate on OPG/RANKL system in differentiated primary human osteoblasts

Alendronate is a well-established treatment for osteoporosis and suppresses bone resorption by a direct effect on osteoclasts and their precursors. The effect of alendronate on osteoclasts is produced, at least in part, by the receptor activator of nuclear factor kappaB ligand (RANKL) and the osteoprotegerin (OPG) synthesized by the osteoblasts. This study analyzes the effect of alendronate in cell viability, alkaline phosphatase (ALP) activity and RANKL and OPG expression in primary human osteoblasts (hOB). Alendronate at concentrations lower than 10⁻⁵ M did not have a toxic effect on hOB in vitro and did not modify the ALP activity at least for 72 h. Alendronate did not change OPG expression in basal, 10% fetal bovine serum (FBS), and vitamin D-treated cultures. Similar results were observed at the protein level. Unexpectedly, alendronate at 10⁻⁷ and 10⁻⁵ M concentrations increased the RANKL expression with the presence of vitamin D in differentiated hOB, and this induction of RANKL mRNA levels by alendronate was dose-dependent. However, this effect was not observed in basal and 10% FBS culture conditions. Thus, we conclude that alendronate does not affect the ALP activity and OPG gene expression in differentiated hOB, but may increase RANKL gene expression induced by vitamin D.

The effect of the alendronate on OPG/RANKL system in differentiated primary human osteoblasts

Alendronate is a well-established treatment for osteoporosis and suppresses bone resorption by a direct effect on osteoclasts and their precursors. The effect of alendronate on osteoclasts is produced, at least in part, by the receptor activator of nuclear factor kappaB ligand (RANKL) and the osteoprotegerin (OPG) synthesized by the osteoblasts. This study analyzes the effect of alendronate in cell viability, phosphatase alkaline (ALP) activity and RANKL, and OPG expression in primary human osteoblasts (hOB). Alendronate at concentrations lower than 10(-5) M did not have a toxic effect on hOB in vitro and did not modify the ALP activity at least for 72 h. Alendronate did not change OPG expression in basal, 10% FBS, and vitamin D-treated cultures. Similar results were observed at the protein level. Unexpectedly, alendronate at 10(-7) and 10(-5) M concentrations increased the RANKL expression with the presence of vitamin D in differentiated hOB and this induction of RANKL mRNA levels by alendronate was dose-dependent. However, this effect was not observed in basal and 10% FBS culture conditions. Thus, we conclude that alendronate does not affect the ALP activity and OPG gene expression in differentiated hOB, but may increase RANKL gene expression induced by vitamin D.

Commitment to the Osteoblast Lineage Is Not Required for RANKL Gene Expression

Differentiation of bone-resorbing osteoclasts from hematopoietic precursors depends upon expression of the cytokine receptor activator of NFkappaB ligand (RANKL) by fibroblastic stromal cells, which some evidence suggests are of the osteoblast lineage. We have shown previously that hormonal-responsiveness of the murine RANKL gene is mediated in part by a distal enhancer that binds Runx2, a transcription factor required for commitment to the osteoblast lineage, supporting the idea that osteoclast-supporting stromal cells may be osteoblasts or their progenitors. However, in this study we demonstrate that parathyroid hormone (PTH) stimulation of RANKL in mice is not affected by a significant reduction in the number of osteoblasts. Consistent with this, neither Runx2, nor Cbfb, a binding partner essential for Runx activity, are required for basal or PTH-stimulated RANKL expression in fibroblastic stromal cell models. Nonetheless, RANKL responsiveness to PTH was elevated in cultured calvaria cells expressing high levels of osterix, another transcription factor required for osteoblast differentiation, and this was associated with elevated PTH receptor expression. The responsiveness of RANKL to 1,25-dihydroxyvitamin D(3) was not elevated in the osterix-expressing cells. Together, these results suggest that commitment to the osteoblast lineage is not a requirement for RANKL gene transcription in fibroblastic stromal cells but may enhance responsiveness of this gene to specific hormones via control of their receptors.