Receptor Activator Of NF-kappaB Ligand mRNA Research Articles

Roles of Dkk2 in the Linkage from Muscle to Bone during Mechanical Unloading in Mice.

Mechanical unloading simultaneously induces muscle and bone loss, but its mechanisms are not fully understood. The interactions between skeletal muscle and bone have been recently noted. Although canonical wingless-related integration site (Wnt)/β-catenin signaling is crucial for bone metabolism, its roles in the muscle and bone interactions have remained unknown. Here, we performed comprehensive DNA microarray analyses to clarify humoral factors linking muscle to bone in response to mechanical unloading and hypergravity with 3 g in mice. We identified Dickkopf (Dkk) 2, a Wnt/β-catenin signaling inhibitor, as a gene whose expression was increased by hindlimb unloading (HU) and reduced by hypergravity in the soleus muscle of mice. HU significantly elevated serum Dkk2 levels and Dkk2 mRNA levels in the soleus muscle of mice whereas hypergravity significantly decreased those Dkk2 levels. In the simple regression analyses, serum Dkk2 levels were negatively and positively related to trabecular bone mineral density and mRNA levels of receptor activator of nuclear factor-kappa B ligand (RANKL) in the tibia of mice, respectively. Moreover, shear stress significantly suppressed Dkk2 mRNA levels in C2C12 cells, and cyclooxygenase inhibitors significantly antagonized the effects of shear stress on Dkk2 expression. On the other hand, Dkk2 suppressed the mRNA levels of osteogenic genes, alkaline phosphatase activity and mineralization, and it increased RANKL mRNA levels in mouse osteoblasts. In conclusion, we showed that muscle and serum Dkk2 levels are positively and negatively regulated during mechanical unloading and hypergravity in mice, respectively. An increase in Dkk2 expression in the skeletal muscle might contribute to disuse- and microgravity-induced bone and muscle loss.

Preliminary investigation on the molecular mechanisms underlying the correlation between VDR-FokI genotype and periodontitis.

The only polymorphism that could change the protein structure in vitamin D receptor (VDR) is the FokI polymorphism (rs2228570). The FF genotype has the strongest transcriptional activity of VDR and is correlated with higher susceptibility to periodontitis. To reveal the possible molecular mechanisms for the correlation preliminarily, the influence of VDR-FokI genotype on the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) in human gingival fibroblasts (hGFs) and human periodontal ligament cells (hPDLCs) was investigated in this study. hGFs and hPDLCs from 15 donors (five FF, seven Ff, and three ff) were treated with 1,25OH2 D3 , with or without the specific knockdown of VDR using siRNA. The mRNA and protein expression of OPG and RANKL were detected using real-time PCR and enzyme-linked immunosorbent assay, respectively. Both in hGFs and hPDLCs, 1,25OH2 D3 could significantly induce the mRNA and protein expression of RANKL, and FF genotype had significantly higher induction than the other genotypes, however, neither 1,25OH2 D3 nor VDR-FokI had significant influence on the OPG expression. As a result, the RANKL/OPG ratio was significantly elevated under 1,25OH2 D3 stimulation and FF genotype had the most remarkable elevation. When VDR was knocked down, all the differences among the three genotypes disappeared. The strongest transcriptional activity of FF genotype might contribute to the strongest enhancement of RANKL expression and RANKL/OPG ratio in hGFs and hPDLCs stimulated by 1,25OH2 D3 , which might help to reveal the mechanisms of the correlation between FF genotype and susceptibility to periodontitis.

Abstract 1970: Melatonin inhibits osteolytic bone metastasis through interrupting cancer cells-derived osteolytic factor and reduce osteoclast differentiation

Abstract Osteolytic bone metastasis is seen in lung, breast, and prostate cancer and is associated with severe complications such as bone pain, fractures and hypercalcemia. In the bone microenvironment, osteoclasts play an important role in bone remodeling and bone resorption. Metastatic cancer cells may increase osteoclast differentiation and activity by releasing inflammatory cytokines including receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and IL-8, leading to bone resorption and osteolytic lesions. The endogenous hormone melatonin is known to modulate bone resorption. Increasing evidence suggests that melatonin inhibits cancer cell proliferation, angiogenesis and metastasis. Up until now, the inhibitory role of melatonin in osteolytic bone metastasis has remained unclear. In In vitro study, we observed that melatonin inhibits the differentiation of precursor osteoclasts and promotes apoptosis in mature osteoclasts. We injected mice tibia with luciferase-labeled osteolytic cancer cell lines (lung cancer: A549, prostate cancer: PC-3) to establish bone metastasis. After 4 weeks of intraperitoneal injection with melatonin, IVIS fluorescence signaling indicated that melatonin inhibited tumor growth. X-ray imaging of the bone revealed an improvement in bone resorption after melatonin treatment. In In vitro melatonin also reduced RANKL protein and mRNA expression in the A549 and PC-3 cell lines. Our data indicate that melatonin improves bone mass by inhibiting the expression of cancer-derived osteolytic factors and by promoting apoptosis in mature osteoclasts. Our future investigations will seek to determine the signaling pathway involved in melatonin-regulated RANKL expression in cancer cells. In summary, our findings provide insight into the benefits of melatonin treatment in osteolytic bone metastasis. Citation Format: Jyun-Lin Lai, An-Chen Chang, Po-Chun Chen, Chih-Hsin Tang. Melatonin inhibits osteolytic bone metastasis through interrupting cancer cells-derived osteolytic factor and reduce osteoclast differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1970.

Effect of TNF-α-Induced Sclerostin on Osteocytes during Orthodontic Tooth Movement.

Osteocytes are abundant cells in bone, which contribute to bone maintenance. Osteocytes express receptor activator of nuclear factor kappa-B ligand (RANKL) and regulate osteoclast formation. Orthodontic tooth movement (OTM) occurs by osteoclast resorption of alveolar bone. Osteocyte-derived RANKL is critical in bone resorption during OTM. Additionally, tumor necrosis factor-α (TNF-α) is important in osteoclastogenesis during OTM. Sclerostin has been reported to enhance RANKL expression in the MLO-Y4 osteocyte-like cell line. This study investigated the effect of TNF-α on sclerostin expression in osteocytes during OTM. In vitro analysis of primary osteocytes, which were isolated from DMP1-Topaz mice by sorting the Topaz variant of GFP-positive cells, revealed that SOST mRNA expression was increased when osteocytes were cultured with TNF-α and that RANKL mRNA expression was increased when osteocytes were cultured with sclerostin. Moreover, the number of TRAP-positive cells was increased in osteocytes and osteoclast precursors cocultured with sclerostin. In vivo analysis of mouse calvariae that had been subcutaneously injected with phosphate-buffered saline (PBS) or TNF-α revealed that the number of TRAP-positive cells and the percentage of sclerostin-positive osteocytes were higher in the TNF-α group than in the PBS group. Furthermore, the level of SOST mRNA was increased by TNF-α. As an OTM model, a Ni-Ti closed-coil spring connecting the upper incisors and upper-left first molar was placed to move the first molar to the mesial direction in wild-type (WT) mice and TNF receptor 1- and 2-deficient (TNFRsKO) mice. After 6 days of OTM, the percentage of sclerostin-positive osteocytes on the compression side of the first molar in TNFRsKO mice was lower than that in WT mice. In this study, TNF-α increased sclerostin expression in osteocytes, and sclerostin enhanced RANKL expression in osteocytes. Thus, TNF-α may play an important role in sclerostin expression in osteocytes and enhance osteoclast formation during OTM.

Expression Profiling of Receptor-Activator of Nuclear Factor-Kappa B Ligand in Soft Tissue Tumors.

Bone and soft tissue tumors are derived from mesenchymal cells, and they are hard to treat. Receptor-activator of nuclear factor-kappa B ligand (RANKL) is an essential cytokine for osteoclast differentiation and activation and is expressed on the surface of osteoblasts or stromal cells. In this study, to explore the potential of denosumab treatment for soft tissue tumors, we analyzed the expression profiles of RANKL mRNA in 425 tumor specimens of 33 histological types by real-time RT-PCR. Denosumab is a monoclonal antibody that prevents the binding of RANKL to receptor-activator of nuclear factor-kappa B (RANK). For comparison, the relative expression levels of RANK and osteoprotegerin (OPG) mRNAs were also measured. OPG functions as a soluble decoy receptor for RANKL. Higher expression levels of RANKL mRNA were detected in calcifying aponeurotic fibroma, fibrosarcoma, calcifying epithelioma, myositis ossificans, heterotopic calcification, giant cell tumor of the tendon sheath (GCTTS), and pigmented villonodular synovitis (PVNS), compared with the levels of other tumor types. Moreover, the expression levels of RANK mRNA were highest in GCTTS, followed by myositis ossificans and PVNS, whereas the expression levels of OPG mRNA were greatly varied among these histological types. We then analyzed RANKL protein expression by immunohistochemistry in 57 tumor specimens with higher expression levels of RANKL mRNA. RANKL-positive cells were detected in GCTTS, PVNS, myositis ossificans, heterotopic calcification, and calcifying aponeurotic fibroma. In conclusion, RANKL is expressed in subsets of soft tissue tumors with calcification, and denosumab is a potential therapeutic option for soft tissue tumors expressing RANKL.

Electroacupuncture Intervention Improves Cartilage Degeneration and Subchondral Bone Osteoporosis of Knee-joint Possibly by Adjusting OPG/RANK/RANKL Signaling in Ovariectomized Rats

To observe the effect of electroacupuncture (EA) on histopathological changes of cartilage and subchondral bone and osteoprotegerin (OPG)，receptor activator of nuclear factor-κB (RANK), and RANK ligand (RANKL) (OPG/RANK/RANKL) signaling and the expression of matrix metalloproteinase-13 (MMP-13) in ovariectomized(OVX)rats, so as to explore its mechanism underlying improvement of osteoporosis. Three-month female SD rats were randomly divided into sham operation, model and EA groups (n＝8 in each group). The ovoariectomy model was established by resection of bilateral ovaries. Rats of the sham group were treated by simple removal of a piece of adipose tissue around the bilateral ovaries. EA (3 Hz/15 Hz, 1 mA) was applied to bilateral "Sanyinjiao" (SP 6), "Yanglingquan" (GB 34), "Yinlingquan" (SP 9) and "Zusanli" (ST 36) for 30 min, once daily (except the weekends) for 12 weeks. The histopathological changes of the subchondral bone of the right knee-joint were observed after Saffron O dyeing and evaluated by Mankin's score, and its anatomical structure including the bone volume fraction (bone volume/tissue volume, BV/TV), trabecular bone number (Tb. N) and trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) was observed by using Micro CT imaging. The urine C-terminal cross-linking telopeptide of type Ⅰ collagen (CTX-Ⅰ), CTX-Ⅱ (two bone resorption markers) and serum estrogen (E 2) contents were assayed by ELISA, and the expression levels of OPG, RANKL and MMP-13 mRNAs in the cartilage tissue of the left knee-joint were detected by quantitative RT-PCR. Following modeling, the BV/TV, Tb. N and Tb.Th levels were significantly decreased (P<0.01) and Tb.Sp and Mankin's score obviously increased in the model group compared with the sham group (P<0.01), suggesting a formation of osteoporosis and degeneration of the cartilage tissue. The serum E 2 content and OPG mRNA level in the cartilagetissue were significantly down-regulated (P<0.01), and urine CTX-Ⅰ and CTX-Ⅱ contents and RANKL mRNA and MMP-13 mRNA expression levels cartilagetissue were considerably up-regulated in the model group relevant to the sham group (P<0.01). After EA intervention, modeling-induced decrease of BV/TV, Tb.N, Tb.Th, E 2 and OPG mRNA levels and OVX-induced increase of Tb.Sp, Mankin's score, CTX-Ⅰ， CTX-Ⅱ， RANKL mRNA and MMP-13 mRNA levels were all completely reversed in the EA group relevant to the model group (P<0.01，P<0.05)．. EA intervention can inhibit subchondral bone osteoporosis and articular cartilage degeneration of knee-joint in OVX rats, which is closely associated with its effects in inhibiting the down-regulation of serum E 2 and OPG mRNA expression and up-regulation of CTX-Ⅰ， CTX-Ⅱ， RANKL mRNA and MMP-13 mRNA levels, including adjusting OPG/RANK/RANKL signaling.

Effect of esculetin on bone metabolism in ovariectomized rats

OBJECTIVETo determine the effect of an esculetin formulation (at 97.4% purity) on osteoporosis, and to investigate the potential underlying molecular mechanism(s). METHODSSixty specific pathogen free-grade female Wistar rats were randomly assigned to three groups: blank control (n = 12), sham (n = 12), and model (n = 36). The model group were bilaterally ovariectomized. The sham group had the tissue surrounding the ovaries removed, while the ovaries were retained. After 3 months, the model group was randomly divided into three subgroups: OVX (n = 12), positive control (n = 12), and esculetin (n = 12). The positive control group and the esculetin group were intragastrically administered diethylstilbestrol (0.046 mg • kg−1 • d−1) or esculetin (384 mg • kg−1 • d−1), respectively, once per day for 6 consecutive days; medication administration was then stopped for 1 d, before being administered for another 6 consecutive days. All rats were treated for 3 months. Samples were collected at the end of the treatment period. An Osteocore3 Digital 2D bone densitometer was used to test the bone mineral density, and histomorphometric analysis was performed to measure bone mass, bone formation, and bone resorption. Enzyme-linked immunosorbent assay analysis was used to measure the serum concentrations of interleukin-6 (IL-6), osteoprotegerin (OPG), and receptor activator of nuclear factor-kappa B ligand (RANKL). Immunohistochemistry and in situ hybridization were performed to detect the protein and mRNA expressions of OPG and RANKL in osteoblasts and bone marrow stromal cells. RESULTSCompared with the OVX group, the esculetin group had significantly greater femoral bone mineral density and tibial trabecular bone volume, and significantly smaller trabecular resorption surface. The percentage of trabecular formation surface, average osteoid width, trabecular bone mineralization rate, and cortical bone mineralization rate did not significantly differ between groups. Compared with the sham group, the esculetin group had significantly decreased serum levels of IL-6 and RANKL, and significant downregulation of RANKL protein and mRNA expression levels in osteoblasts and bone marrow stromal cells; however, there was no significant difference between groups in OPG. CONCLUSIONEsculetin can increase bone mass by upregulating RANKL expression in osteoblasts and bone marrow stromal cells, and decreasing serum IL-6 concentration. This indicates that the therapeutic effect of esculetin on osteoporosis occurs via decreased bone resorption.

TRPM3/TRPV4 regulates Ca2+-mediated RANKL/NFATc1 expression in osteoblasts

Mechanical stress plays an important role in the regulation of bone turnover. However, the mechanism underlying hypo-osmotic stress-induced cellular response in osteoblasts remains poorly understood. In this study, we investigated the effect of hypotonic stress on the expression of bone remodeling factors, including the receptor activator of nuclear factor-kappa B ligand (RANKL) and the nuclear factor of activated T cells type c1 (NFATc1) in primary mouse osteoblasts and MC3T3-E1 cells. Hypo-osmotic stress induced significant increases in RANKL mRNA expression and intracellular Ca2+ concentration ([Ca2+]i) from the extracellular space. Hypo-osmotic stress-induced effects on [Ca2+]i and RANKL and NFATc1 protein expression were decreased by antagonists of transient receptor potential melastatin 3 (TRPM3) and vanilloid 4 (TRPV4). Agonists of TRPM3 and TRPV4 activated [Ca2+]i and RANKL and NFATc1 protein expression. Furthermore, genetic suppression of Trpm3 and Trpv4 reduced hypo-osmotic stress-induced effects in mouse osteoblasts. These results suggest that hypo-osmotic stress induces increases in [Ca2+]i through TRPM3 and TRPV4 to regulate RANKL and NFATc1 expression in mouse osteoblastic cells and that mechanical stress-activated TRP channels may play a critical role in bone remodeling.

MiR-302a-3p regulates RANKL expression in human mandibular osteoblast-like cells.

Receptor activator of nuclear factor kappa-B ligand (RANKL) is important substance during osteoclastogenesis that resulted in alveolar bone loss of periodontitis. MicroRNAs (miRNAs) regulate gene expression in several biological processes including osteoclastogenesis. We investigated the function of microRNA-302a-3p (miR-302a-3p) to regulate receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human mandibular osteoblast-like cells (HMOBs). HMOBs were incubated with prostaglandin E2 (PGE2 ) to mimic inflammation, or with PGE2 and interferon gamma (IFNγ) to mimic homeostasis. MicroRNA (miRNA) profiles related to RANKL expression were demonstrated by PCR array, and miR-302a-3p was identified. Using TargetScanHuman 7.0, a target of miR-302a-3p was predicted. To confirm its function, miR-302a-3p was overexpressed, or silenced, by transfection with miR-302a-3p mimic, or inhibitor, respectively. Level of miR-302a-3p and RANKL mRNA was assessed by qRT-PCR. Soluble RANKL (sRANKL), and membrane-bound RANKL (mRANKL) were measured by ELISA and by Western blot, respectively. When PGE2 stimulated RANKL in HMOBs, miR-302a-3p was lower than baseline level. However, upregulation of miR-302a-3p is observed when IFNγ suppressed RANKL expression in PGE2 -stimulated HMOBs. miR-302a-3p was predicted to target PRKACB mRNA encoding the catalytic subunit in cAMP/PKA pathway. Overexpression of miR-302a-3p could decrease RANKL expression during PGE2 stimulation. In contrast, silencing of miR-302a-3p by its inhibitor increased RANKL expression in PGE2 -IFNγ conditioned HMOBs. miR-302a-3p regulates RANKL expression in HMOBs within PGE2 -IFNγ regulatory network.

Effect of hypoxia on the expression of CCAAT/enhancer-binding protein β and receptor activator of nuclear factor kappa-B ligand in periodontal ligament cells.

Hypoxia after traumatic injuries to a tooth is one of the causes of subsequent root resorption. Inflammatory cytokines produced under hypoxic conditions are associated with root resorption, but the mechanism has not been fully understood. In this study, the role of hypoxia-inducible factor-1 (HIF-1) signaling in the regulation of CCAAT (cytosine-cytosine-adenosine-adenosine-thymidine)/enhancer-binding protein-β (C/EBPβ) and the receptor activator of nuclear factor kappa-B ligand (RANKL) expressions in immortalized human periodontal ligament (PDL) cells was investigated. PDL cells cultured under a hypoxic condition showed an increase in the expression of C/EBPβ and RANKL messenger RNAs (mRNAs), whereas the expression of osteoprotegerin and HIF-1α mRNAs was unaffected. Hypoxia had no effects on the secretion of interleukin (IL)-1β, IL-6, IL-8, IL-17A, tumor necrosis factor-alpha, macrophage migration inhibitory factor, monocyte chemoattractant protein-1, and macrophage colony-stimulating factor in the culture media. Treatment of the cells with dimethyloxaloylglycine, a competitive HIF prolyl hydroxylase inhibitor, significantly increased the expression of C/EBPβ and RANKL mRNAs. This suggested that the hypoxia-induced elevation of C/EBPβ and RANKL mRNAs was dependent on the HIF-1 activity. PDL cells transfected with a specific small interfering RNA designed to target the C/EBPβ gene showed a significant suppression of the RANKL mRNA. These findings indicated that C/EBPβ may play an important role in tooth root resorption via RANKL activation in hypoxia-exposed PDL cells.

A study on OPG/RANK/RANKL axis in osteoporotic bile duct-ligated rats and the involvement of nitrergic and opioidergic systems.

Chronic liver disease (CLD) affects millions of people and its impact on bone loss has become a subject of interest. Nitric oxide and endogenous opioids are suggested to increase during cholestasis/cirrhosis and may impact bone resorption by different mechanisms. The receptor activator of nuclear factor-κB (RANK)/RANK-ligand (RANKL)/osteoprotegerin (OPG) signaling pathway regulates bone resorption, but its role in metabolic bone disease subsequent to CLD is unknown. We aimed to investigate the involvement of nitrergic and opioidergic systems in bone loss relative to the RANK/RANKL/OPG pathway, in bile duct-ligated (BDL) rats. Eighty BDL/sham-operated (SO) rats received injections of 3 mg/kg/day Nω-Nitro-L-arginine methyl ester ± naltrexone (10 mg/kg/day) or saline for 28 days. Plasma bone turnover markers, OPG, RANK, and RANKL along with mRNA expression levels of the latter three were assessed. Plasma bone turnover markers and OPG level increased, but RANKL decreased in the BDL group compared with their SO controls (both: P ≤ 0.05). Administration of naltrexone reduced bone turnover markers and OPG level while increased RANKL content in comparison to BDL rats (P ≤ 0.05). As compared to untreated BDL rats, nitric oxide inhibition showed no effect on bone turnover marker i.e. OPG, RANK, and RANKL levels. BDL significantly increased RANK mRNA, but had no significant effect on RANKL and OPG mRNA expression. The lack of association between plasma levels and quantitative gene expression of RANKL and OPG suggests an indirect function of these markers in BDL rats. Considering that opioid receptor blockage by naltrexone in BDL animals caused a significant decrease in OPG and an increase in RANKL plasma contents, it could be postulated that the opioidergic system may have a regulatory effect on these bone markers.

P38 MAPK and Its Upstream Activators MKK3 and MKK6 Play an Important Role on Lipopolysaccharide-Induced RANKL Expression by Periodontal Ligament Cells

Resident, non-immune cells express various pattern-recognition receptors and produce inflammatory cytokines in response to microbial antigens, during the innate immune response. Alveolar bone resorption is the hallmark of destructive periodontitis and it is caused by the host response to bacteria and their mediators present on the biofilm. The balance between the expression levels of receptor activator of nuclear factorkappa B ligand (RANKL) and osteoprotegerin (OPG) is pivotal for osteoclast differentiation and activity and has been implicated in the progression of bone loss in periodontitis. To assess the contribution of resident cells to the bone resorption mediated by innate immune signaling, we stimulated fibroblasts and osteoblastic cells with LPS from. Escherichia coli (TLR4 agonist), Porphyromonas gingivalis (TLR2 and -4 agonist), and interleukin-1 beta (as a control for cytokine signaling through Toll/IL-1receptor domain) in time-response experiments. Expression of RANKL and OPG mRNA was studied by RT-PCR, whereas the production of RANKL protein and the activation of p38 MAPK and NF-kB signaling pathways were analyzed by western blot. We used biochemical inhibitors to assess the relative contribution of p38 MAPK and NF-kB signaling to the expression of RANKL and OPG induced by TLR2, -4 and IL1β in these cells. Both p38 MAPK and NFkB pathways were activated by these stimuli in fibroblasts and osteoblasts, but the kinetics of this activation varied in each cell type and with the nature of the stimulation. E. coli LPS was a stronger inducer of RANKL mRNA in fibroblasts, whereas LPS from P. gingivalis downregulated RANKL mRNA in periodontal ligament cells but increased its expression in osteoblasts. IL-1β induced RANKL in both cell types and without a marked effect on OPG expression. p38 MAPK was more relevant than NF-kB for the expression of RANKL and OPG in these cell types.

Ameloblastin and enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway

Ameloblastin (Ambn) and enamelin (Enam) play a pivotal role in enamel mineralization. Previous studies have demonstrated that these enamel-related gene products also affect bone growth and remodeling; however, the underlying mechanisms have not been elucidated. In the present study, we examined the effects of Ambn and Enam on the receptor activator of nuclear factor kappa-B ligand (RANKL) expression induced with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone (DEX) on mouse bone marrow stromal cell line ST2 cells. We then verified the effect of Ambn and Enam on osteoclastogenesis. We found that pretreatment with recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) remarkably suppressed RANKL mRNA and protein expression induced with 1,25(OH)2D3 and DEX. Interestingly, rhAmbn and rhEnam attenuated the phosphorylation of mitogen-activated protein kinases (MAPK), including ERK1/2, JNK, and p38 in ST2 cells stimulated with 1,25(OH)2D3 and DEX. Moreover, pretreatment with specific inhibitors of ERK1/2 and p38, but not JNK, blocked RANKL mRNA and protein expression. Cell co-culture results showed that rhAmbn and rhEnam downregulated mouse bone marrow cell differentiation into osteoclasts induced with 1,25(OH)2D3 and DEX-stimulated ST2 cells. These results suggest that Ambn and Enam may indirectly suppress RANKL-induced osteoclastogenesis via downregulation of p38 and ERK1/2 MAPK signaling pathways in bone marrow stromal cells.

Low-intensity pulsed ultrasound inhibits lipopolysaccharide-induced IL-6 and RANKL expression in osteoblasts.

Periodontal disease is caused by inflammation induced by Porphyromonas gingivalis (P.g.) lipopolysaccharide (LPS) and involves expression of proinflammatory cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor-α, and receptor activator of nuclear factor kappa B ligand (RANKL), which are implicated in bone resorption. Low-intensity pulsed ultrasound (LIPUS) is commonly used in the treatment of bone fracture. However, the mechanisms by which LIPUS inhibits LPS-induced inflammatory cytokines are poorly understood. Therefore, we investigated the effects of LIPUS on LPS-induced expression of the proinflammatory cytokines IL-6 and RANKL. MC3T3-E1 cells were incubated in the presence or absence of P.g. LPS and then stimulated with LIPUS for 30 min/day for a maximum of 14 days. LPS increased mRNA and protein expressions of IL-6 and RANKL on day 14. In addition, mRNA expression of COX-2 LPS was higher after 3 and 7 days of LIPUS treatment. PGE2 was induced by LPS after 7 and 14 days of culture. LIPUS suppressed all stimulatory effects of LPS. These results suggest that LIPUS inhibits LPS-induced expression of inflammation cytokines by suppressing PGE2 production and might thus have potential applications in the treatment of periodontitis.

Silibinin inhibitis progesterone induced RANKL expression in human uterine leiomyoma cells

Silibinin is a natural compound that was reported to inhibit the effects of RANKL; the underlying mechanism however is unclear. We previously demonstrated that progesterone stimulates receptor activator of nuclear factor kappa-B ligand (RANKL) expression in leiomyoma. Here we investigate whether silibinin inhibits progesterone induced RANKL expression, cell cycle proteins and extracellular matrix deposition. Molecular analysis in freshly isolated leiomyoma tissue and cells. Fresh leiomyoma tissue explants (n= 3 patients) were treated with vehicle or silibinin alone, as well as in the presence or absence of synthetic progesterone agonist R5020. Total RNA was extracted and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to asses fold change of RANKL mRNA levels. Next, we isolated leiomyoma smooth muscle cells from fresh fibroid tissues and maintained them in primary culture (n=3). The cells were treated with vehicle or varying doses of silibinin (30, 60, or 90 mM) for 24, 48, or 72 hours (h) respectively. Total RNA was extracted and RT-PCR was used to measure mRNA levels of RANKL, Cyclin D1 (a downstream target of RANKL and a cell cycle protein increased with cell proliferation) and Col1A2 and Col3A1 (markers of extracellular matrix deposition). Treatment of tissue explants with R5020 led to a significant increase in RANKL expression (p<0.001), which was significantly decreased by treatment with silibinin (p<0.01). In primary cells, silibinin reduced RANKL expression in a dose-dependent manner with the most significant change observed at 60 mM (up to 60% decrease, p<0.05). Silibinin significantly decreased cyclin D1, Col1A2 and Col3A1 mRNA levels in a dose-dependent manner by up to 80% change (p<0.05). Inhibition of RANKL, Cyclin D1, Col1A2 and Col3A1 expression by silibinin was observed as early as 24 h, peaked at 48 h and persisted till 72 h of treatment. Silibinin is an effective inhibitor of RANKL expression. Silibinin also decreased proliferative activity and extracellular matrix deposition in leiomyoma smooth muscle cells. Thus, silibinin may serve as a novel medical treatment option with no known side effects for uterine leiomyoma.

Drug-releasing nano-engineered titanium implants: therapeutic efficacy in 3D cell culture model, controlled release and stability

There is an ongoing demand for new approaches for treating localized bone pathologies. Here we propose a new strategy for treatment of such conditions, via local delivery of hormones/drugs to the trauma site using drug releasing nano-engineered implants. The proposed implants were prepared in the form of small Ti wires/needles with a nano-engineered oxide layer composed of array of titania nanotubes (TNTs). TNTs implants were inserted into a 3D collagen gel matrix containing human osteoblast-like, and the results confirmed cell migration onto the implants and their attachment and spread. To investigate therapeutic efficacy, TNTs/Ti wires loaded with parathyroid hormone (PTH), an approved anabolic therapeutic for the treatment of severe bone fractures, were inserted into 3D gels containing osteoblast-like cells. Gene expression studies revealed a suppression of SOST (sclerostin) and an increase in RANKL (receptor activator of nuclear factor kappa-B ligand) mRNA expression, confirming the release of PTH from TNTs at concentrations sufficient to alter cell function. The performance of the TNTs wire implants using an example of a drug needed at relatively higher concentrations, the anti-inflammatory drug indomethacin, is also demonstrated. Finally, the mechanical stability of the prepared implants was tested by their insertion into bovine trabecular bone cores ex vivo followed by retrieval, which confirmed the robustness of the TNT structures. This study provides proof of principle for the suitability of the TNT/Ti wire implants for localized bone therapy, which can be customized to cater for specific therapeutic requirements.

Jagged1 inhibits osteoprotegerin expression by human periodontal ligament cells

Notch signaling regulates bone homeostasis. The present study investigated the effect of Jagged1 on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human periodontal ligament stromal (hPDL) cells. hPDL cells were seeded on to indirect immobilized Jagged1 surfaces. OPG expression was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Lentiviral small hairpin RNA particles against NOTCH2 were employed to inhibit NOTCH2 expression. Osteoclast formation was evaluated using RAW264.7 cells. An influence of exogenous OPG on osteogenic differentiation was determined by real-time polymerase chain reaction and Alizarin Red S staining. Jagged1 significantly enhanced HES1 and HEY1mRNA expression in a dose-dependent manner. Furthermore, OPG mRNA and protein levels dramatically decreased upon exposing hPDL cells to Jagged1. However, RANKL mRNA levels were not significantly different. There was also no difference in M-CSF and MCP-1mRNA expression. A γ-secretase inhibitor and cycloheximide treatment rescued Jagged1-attenuated OPG expression. Furthermore, shNOTCH2 overexpressing hPDL cells did not exhibit a decrease in OPG expression upon exposure to Jagged1, implying the involvement of NOTCH2 in the regulatory mechanism. Culturing RAW264.7 cells with conditioned medium from Jagged1-treated hPDL cells enhanced osteoclast formation compared with those cultured with conditioned medium of the control group. Lastly, OPG treatment did not influence osteogenic differentiation by hPDL cells. These results suggest that Jagged1 activates Notch signaling in hPDL cells, leading to decreased OPG expression. This may imply an indirect role of Jagged1 on the regulation of osteoclast differentiation via hPDL cells.

Interleukin-17 is involved in orthodontically induced inflammatory root resorption in dental pulp cells.

The objectives of this study were (1) to investigate the expressions of interleukin (IL)-17, RANKL (the receptor activator of NF-kappaB ligand), and osteoprotegerin (OPG) in root resorption areas during experimental tooth movement in rats, and (2) to determine the effect of IL-17 on the expressions of RANKL and OPG mRNA from human dental pulp cells. Twelve male 6-week-old Wistar rats were subjected to an orthodontic force of 50 g to induce a mesially tipping movement of the maxillary first molars for 7 days. The expression levels of tartrate resistant acid phosphatase (TRAP), interleukin (IL)-17, IL-17 receptor (IL-17R), receptor activator of nuclear factor-kappa B ligand (RANKL), and OPG proteins were determined in dental pulp by immunohistochemical analysis. Furthermore, the effects of IL-17 on the expressions of RANKL and OPG mRNA were investigated using human dental pulp cells in vitro. In the experimental tooth movements in vivo, resorption lacunae with multinucleated cells were observed in the 50-g group. The immunoreactivities for IL-17, IL-17R, and RANKL were detected in dental pulp tissues subjected to the orthodontic force on day 7. Moreover, IL-17 increased the mRNA expression of RANKL from human dental pulp cells in vitro. The results of this study suggest that IL-17 and RANKL may be involved in the process of orthodontically induced inflammatory root resorption in dental pulp cells.

Downregulation of RANKL and RANKL/osteoprotegerin ratio in human periodontal ligament cells during their osteogenic differentiation.

Human periodontal ligament cells (hPDLCs) are considered the promising seed cells in periodontal tissue engineering. Previous studies have discovered the ability of hPDLCs in alveolar bone formation. It remains unclear, however, how the expression of factors associated with osteoclastogenesis in hPDLCs change during their osteogenic differentiation. The present study aimed to observe the regulation of receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) in hPDLCs during their osteogenic differentiation. hPDLCs were treated with (M group) or without (C group) the osteogenic induction medium. Alkaline phosphatase activity was displayed with the Gomori calcium phosphate method. Mineralized nodules were detected with von Kossa staining. Expression levels of RANKL and OPG in hPDLCs were analyzed with real-time reverse transcription-polymerase chain reaction and western blot. Tartrate-resistant acid phosphatase (TRAP) staining was used to display the TRAP activity in Raw264.7 cells co-cultured with hPDLCs in the M group and the C group. We found that alkaline phosphatase staining was shown to be remarkably higher in the M group than that in the C group during the 21 d interval. Mineralized nodules could be seen in the M group but not in the C group. The expression levels of RANKL mRNA significantly decreased in the M group by 1.69-fold (p = 0.096) at day 7, by 2.04-fold (p = 0.000) at day 14 and by 1.84-fold (p = 0.023) at day 21, compared with the corresponding levels of RANKL in the C group. Similarly, the levels of RANKL protein decreased in the M group by 1.82-fold (p = 0.062) at day 7, by 5.64-fold (p = 0.000) at day 14 and by 4.84-fold (p = 0.000) at day 21. The mRNA and protein expression levels of OPG tended to increase in the M group. As a result, the RANKL/OPG mRNA and protein ratios were significantly downregulated by osteogenic induction. In addition, the number of TRAP staining-positive multinuclear cells in the M group was significantly less than in the C group (p = 0.018). hPDLCs may help inhibit the resorption of alveolar bone during their osteogenic differentiation by reducing the RANKL expression and the RANKL/OPG ratio.

In vitro evaluation of the biological effect of SOFAT on osteoblasts.

Osteoclastogenesis is regulated by osteoblasts especially through the production of receptor activator of nuclear factor kappa-B ligand (RANKL). Immune cells present in inflamed tissues markedly increase this process by upregulating RANKL directly or by secreting proinflammatory cytokines, which stimulate RANKL expression by osteoblasts. A novel T-cell-secreted cytokine, termed secreted osteoclastogenic factor of activated T cells (SOFAT) was recently described. To better understand how SOFAT affects bone metabolism, we investigated its effect on osteoblastic cells. We demonstrate here that SOFAT did not influence MC3T3 cells viability and proliferation, evaluated by trypan blue exclusion and MTT tests, respectively. SOFAT stimulated the secretion of IL-6, IL-10 and GM-CSF in MC3T3 cells, as shown by the analysis of an inflammatory cytokines ELISA array. The upregulation of the corresponding genes was checked by qPCR. Both RANKL mRNA and protein levels did not significantly change in the presence of SOFAT, evaluated by qPCR and western blotting, respectively. In addition, analysis of a PCR array for IL6/STAT3 pathway demonstrated that SOFAT induced the expression of BCL2, IL1B, IL10, IL22, IL2RA, IL4, IL6, TNFSF10 and PIAS3, while IL2, IL21, CD4, CSF3R and TNF were repressed. Our results confirm that the SOFAT mechanism of action is RANKL-independent and indicate that, by co-opting osteoblasts to increase the production of osteoclastogenic cytokines, SOFAT may exacerbate inflammation and support osteoclast formation and bone destruction.