RANKL mRNA Research Articles

Mapping RANKL- and OPG-expressing cells in bone tissue: the bone surface cells as activators of osteoclastogenesis and promoters of the denosumab rebound effect.

Denosumab is a monoclonal anti-RANKL antibody that inhibits bone resorption, increases bone mass, and reduces fracture risk. Denosumab discontinuation causes an extensive wave of rebound resorption, but the cellular mechanisms remain poorly characterized. We utilized in situ hybridization (ISH) as a direct approach to identify the cells that activate osteoclastogenesis through the RANKL/OPG pathway. ISH was performed across species, skeletal sites, and following recombinant OPG (OPG:Fc) and parathyroid hormone 1-34 (PTH) treatment of mice. OPG:Fc treatment in mice induced an increased expression of RANKL mRNA mainly in trabecular, but not endocortical bone surface cells. Additionally, a decreased expression of OPG mRNA was detected in bone surface cells and osteocytes of both compartments. A similar but more pronounced effect on RANKL and OPG expression was seen one hour after PTH treatment. These findings suggest that bone surface cells and osteocytes conjointly regulate the activation of osteoclastogenesis, and that OPG:Fc treatment induces a local accumulation of osteoclastogenic activation sites, ready to recruit and activate osteoclasts upon treatment discontinuation. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data from murine bone marrow stromal cells revealed that Tnfsf11+ cells expressed high levels of Mmp13, Limch1, and Wif1, confirming their osteoprogenitor status. ISH confirmed co-expression of Mmp13 and Tnfsf11 in bone surface cells of both vehicle- and OPG:Fc-treated mice. Under physiological conditions of human/mouse bone, RANKL is expressed mainly by osteoprogenitors proximate to the osteoclasts, while OPG is expressed mainly by osteocytes and bone-forming osteoblasts.

Bone marrow adipogenic lineage precursors are the major regulator of bone resorption in adult mice.

Bone resorption by osteoclasts is a critical step in bone remodeling, a process important for maintaining bone homeostasis and repairing injured bone. We previously identified a bone marrow mesenchymal subpopulation, marrow adipogenic lineage precursors (MALPs), and showed that its production of RANKL stimulates bone resorption in young mice using Adipoq-Cre. To exclude developmental defects and to investigate the role of MALPs-derived RANKL in adult bone, we generated inducible reporter mice (Adipoq-CreER Tomato) and RANKL deficient mice (Adipoq-CreER RANKLflox/flox, iCKO). Single cell-RNA sequencing data analysis, lineage tracing, and in situ hybridization revealed that Adipoq+ cells contain not only MALPs but also late mesenchymal progenitors capable of osteogenic differentiation. However, RANKLmRNA was only detected in MALPs, but not in osteogenic cells. RANKL deficiency in MALPs induced at 3 months of age rapidly increased trabecular bone mass in long bones as well as vertebrae within 1 month due to diminished bone resorption but had no effect on the cortical bone. Ovariectomy (OVX) induced trabecular bone loss at both sites. RANKL depletion either before OVX or at 6 weeks post OVX protected and restored trabecular bone mass. Furthermore, bone healing after drill-hole injury was delayed in iCKO mice. Together, our findings demonstrate that MALPs play a dominant role in controlling trabecular bone resorption and that RANKL from MALPs is essential for trabecular bone turnover in adult bone homeostasis, postmenopausal bone loss, and injury repair.

Role of OPG/RANKL/RANK/TLR4 signaling pathway in sepsis-associated acute kidney injury

BackgroundSepsis-associated acute kidney injury (SA-AKI) has high mortality rates. The osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of nuclear factor-κB (RANK)/Toll-like receptor 4 (TLR4) pathway and its potential role in SA-AKI pathogenesis remain to be fully understood. Herein, we addressed this issue using mouse models.MethodsAn SA-AKI mouse model was established using the cecal ligation and puncture method (CLP). Mice were grouped into sham, CLP model, CLP + recombinant RANKL, and CLP + anti-RANKL groups. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured to assess kidney function. ELISA was used to detect serum IL-1β, TNF-α, and IL-6 levels. Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression levels of OPG, RANKL, RANK, and TLR4 in kidney tissues. HE staining was performed to evaluate the pathological changes.ResultsThe CLP model group showed higher levels of Scr and BUN, indicating impaired kidney function in SA-AKI, compared to the sham group. Treatment with recombinant RANKL in the CLP + recombinant RANKL group reduced Scr and BUN levels, while anti-RANKL treatment in the CLP + anti-RANKL group elevated their levels. Moreover, the CLP model group had significantly increased IL-1β, TNF-α, and IL-6 than the sham group, indicating elevated inflammation in SA-AKI. The CLP + recombinant RANKL group demonstrated decreased cytokine levels, whereas the CLP + anti-RANKL group showed an increase. Additionally, the histopathological evaluation revealed distinct kidney tissue damage in the CLP model group. Recombinant RANKL treatment reduced this damage, while anti-RANKL treatment exacerbated it. Mechanically, the mRNA and protein expression of RANKL were significantly decreased, while those of OPG, RANK, and TLR4 were significantly increased in the CLP model group and the CLP + anti-RANKL group. Interestingly, treatment with recombinant RANKL reversed these changes, as evidenced by significantly increased RANKL but decreased OPG, RANK, and TLR4.ConclusionThe OPG/RANKL/RANK/TLR4 pathway is involved in SA-AKI pathogenesis. Recombinant RANKL treatment attenuates the inflammatory response and kidney tissue damage in SA-AKI, possibly via regulating this pathway. This pathway shows promise as a therapeutic target for SA-AKI.

MMP-7, RANKL mRNA expression in the gingival tissue correlates with periodontitis: An in vivo study

Introduction MMP-7 supports the immune response and can have both beneficial and destructive effects. As part of the innate host defense, MMP-7 is connected to the mucosal antimicrobial defense. Even though research on other Matrix metalloproteinase (MMPs) is well-established, understanding MMP-7 expression is required to establish an improved diagnostic strategy. This research investigates the mRNA expression of MMP-7 and RANKL by RT-qPCR assay. Methods and material Twelve male Sprague Dawley rats were allocated into three groups, no treatment, experimental (7 and 14 days). Periodontitis is induced by sterile wire insertion (0.2 mm) and Enterococcus faecalis inoculation within the gingival sulcus situated between the maxillary right 1st and 2nd molar tooth regions. Following euthanasia, tissue samples from the maxillary gingiva and maxillary jaw were extracted for quantitative real-time PCR assay and histopathological assessment. Results Results showed that at 7 days, there was significant upregulation of MMP-7, which was downregulated in 14 days, as well as migration of the junctional epithelium, attachment loss, inflammatory cells, and fibroblasts, as observed by histological analysis. Conclusions Thus, the study suggests that MMP-7 is associated with the progression of periodontitis.

Correlation between PD-1/PD-L1 and RANKL/OPG in chronic apical periodontitis model of Sprague-Dawley rats.

Chronic apical periodontitis (CAP) is characterized by inflammation and destruction of the apical periodontium that is of pulpal origin, appearing as an apical radiolucent area, and does not produce clinical symptoms. Little is known about whether the PD-1/PD-L1 ratio is associated with the balance between RANKL and OPG in CAP. The relationship between PD-1/PD-L1 and RANKL/OPG in CAP is investigated in this study. A CAP rat model was established using Sprague-Dawley rats. The pulp chambers were exposed to the oral cavity to allow bacterial contamination. The apical tissues of the bilateral mandibular first molars were analyzed for histological morphology using hematoxylin and eosin (H&E) staining. Immunohistochemistry and qRT-PCR were used to determine the expression of PD-1, PD-L1, OPG, and RANKL mRNA and proteins in periapical tissues and mandibular samples, respectively. The radiological images indicated a poorly defined low-density shadow and alveolar bone resorption after periodontitis induction. Histological analysis revealed an infiltration of inflammatory cells and alveolar bone resorption in the periapical tissues. Mandibular mRNA and periapical protein expression of PD-1, PD-L1, and RANKL was upregulated 7-28days after periodontitis induction, while the expression of OPG was downregulated. No significant relationship was observed between PD-1/PD-L1 and RANKL/OPG at either mRNA or protein levels in CAP. There is an increased expression of PD-1, PD-L1, and RANKL and a decreased expression of OPG, indicating progression of CAP.

Effects of fine particulate matter on bone mass and osteogenic differentiation of bone marrow mesenchymal stem cells in mice

To observe the effects of exposure to fine particulate matter(PM_(2.5)) on bone mass, microstructure, biomechanical properties, and osteogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs) in mice. A total of 16 C57BL/6J mice aged 8 weeks were randomly divided into control group(NS group) and PM_(2.5) exposure group(PM group). NS group was given normal saline, PM group was given 14 mg/kg PM_(2.5) suspension, 50 μL, poisoning every 3 day. After 10 weeks, the lungs of mice were taken for HE staining, and the left tibia was taken for Micro CT detection to analyze parameters related to cancellous and cortical bone. The right tibia was taken for HE staining to observe changes in bone trabeculae. Immunohistochemical staining was used to detect type I collagen(Col I), osteoprotegerin(OPG), and nuclear factor-κB receptor activating factor ligand(RANKL) protein expression, tartrate resistant acid phosphatase(TRAP) staining for detection of osteoclasts. Extract primary BMSCs from bilateral femurs, induce osteogenesis, and then perform alkaline phosphatase(ALP) staining to detect ALP activity, alizarin red staining to detect bone mineralization ability, real-time PCR to detect osteocalcin(OCN), ALP, OPG, and RANKL mRNA expression, and biomechanical testing to test the mechanical properties of the femur. Compared with the NS group, the pulmonary alveolar structure of the PM group mice was disrupted and a large number of inflammatory cells gathered. Prompt for successful PM_(2.5) poisoning operation. Micro CT result showed that the bone mineral density(BMD) and bone volume fraction(BV/TV) of the PM group mice were 276.959±15.152 mg/cm~3 and 0.208%±0.009%, respectively. The NS group had 316.709±28.205 mg/cm~3 and 0.236%±0.019%, respectively. The PM group was lower than the NS group(P<0.05), but the trabecular number(Tb. N) There was no statistically significant difference in parameters such as trabecular thickness(Tb. Th) and trabecular separation(Tb. SP)(P>0.05). The HE staining result of the tibia showed that the trabeculae in the NS group were thick, dense, and uniform. The bone trabeculae in the PM group were slender, with a decrease in number, widened spacing, and sparse arrangement. The expression of Col I(0.023±0.009) and OPG(0.036±0.010) in the PM group increased compared to the NS group(0.079±0.007, 0.059±0.012), while the expression of RANKL(0.036±0.006) decreased compared to the NS group(0.022±0.002)(P<0.05); The number of TRAP positive particles increased in the PM group. The experimental result after osteoinduction of BMSCs in mice showed that compared with the NS group, the PM group had a decrease in the number of ALP positive cells and a decrease in the number of calcium nodules. The relative expression of ALP, OCN, and OPG mRNA in the PM group(0.375±0.021, 0.585±0.088, 0.768±0.112) was significantly reduced compared to the NS group(1.001±0.043, 1.006±0.132, 1.002±0.086), while the relative expression of RANKL mRNA(1.278±0.118) was increased compared to the NS group(1.001±0.057)(P<0.05). The biomechanical experimental result showed that the maximum deflection of the NS group was 0.337±0.031 mm, while the maximum deflection of the PM group was 0.258±0.041 mm. Compared with the NS group, the maximum deflection of the PM group decreased significantly(P<0.05), and the maximum stress and maximum load showed a decreasing trend, but the difference was not statistically significant(P>0.05). After 10 weeks of exposure to PM_(2.5), it can affect the bone health of mice, and its mechanism may be related to increased osteoclast activity and inhibition of the osteogenic differentiation ability of BMSCs.

Jaw Bone Invasion of Oral Squamous Cell Carcinoma Is Associated with Osteoclast Count and Expression of Its Regulating Proteins in Patients and Organoids.

Oral squamous cell carcinoma (OSCC) frequently invades the jaw. The exact mechanism of bone invasion remains unclear. This study investigates (premature) osteoclasts and the expression of its differentiation regulating proteins RANKL, OPG and RANK in patients with OSCC. Resection specimens from OSCC patients were divided into NI group (No Invasion), E group (Erosion) or I group (bone Invasion). Tissue sections were stained with Cathepsin K (osteoclast-counting), RANKL, OPG and RANK. The staining intensity was scored on different regions of the tumor: front, center, back and normal mucosa. Immunohistochemistry and qPCR for RANKL/OPG/RANK were performed on five head and neck squamous cell carcinoma (HNSCC) organoids. The mean number of osteoclasts (I group) and premature osteoclasts (E group) was significantly higher compared to the NI group (p = 0.003, p = 0.036). RANKL expression was significantly higher in the tumor front and tumor center compared to normal mucosa (all groups). In the I group, RANKL and RANK expression was significantly higher in the tumor front compared to the tumor back and there was a trend of higher RANKL expression in the tumor front compared to the E group and NI group. qPCR showed a 20-43 times higher RANKL mRNA expression in three out of five tumor organoids compared to a normal squamous cell organoid line. There was no correlation between protein and mRNA expression in the HNSCC organoids. These findings suggest that OSCCs induce bone invasion by stimulating osteoclast activation by regulating the production of RANKL and RANK proteins.

Azilsartan inhibits inflammation-triggered bone resorption and osteoclastogenesis in vivo via suppression of TNF-α expression in macrophages.

Hypertension is a major risk factor for cardiovascular disease (CVD) and is associated with increased bone loss due to excessive activity of the local renin-angiotensin system (RAS). Angiotensinogen/Angiotensin (ANG) II/Angiotensin II type 1 receptor (AT1R) axis is considered as the core axis regulating RAS activity. Azilsartan is an FDA-approved selective AT1R antagonist that is used to treat hypertension. This study aimed to determine whether azilsartan affects formation of osteoclast, resorption of bone, and the expression of cytokines linked with osteoclastogenesis during lipopolysaccharide (LPS)-triggered inflammation in vivo. In vivo, following a 5-day supracalvarial injection of LPS or tumor necrosis factor-alpha (TNF-α) with or without azilsartan, the proportion of bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, which are identified as osteoclasts on mice calvariae were counted. The mRNA expression levels of TRAP, cathepsin K, receptor activator of NF-κB ligand (RANKL), and TNF-α were also evaluated. In vitro, the effect of azilsartan (0, 0.01, 0.1, 1, and 10 μM) on RANKL and TNF-α-triggered osteoclastogenesis were investigated. Also, whether azilsartan restrains LPS-triggered TNF-α mRNA and protein expression in macrophages and RANKL expression in osteoblasts were assessed. Furthermore, western blotting for analysis of mitogen-activated protein kinases (MAPKs) signaling was conducted. Azilsartan-treated calvariae exhibited significantly lower bone resorption and osteoclastogenesis than those treated with LPS alone. In vivo, LPS with azilsartan administration resulted in lower levels of receptor activator of RANKL and TNF-α mRNA expression than LPS administration alone. Nevertheless, azilsartan did not show inhibitory effect on RANKL- and TNF-α-triggered osteoclastogenesis in vitro. Compared to macrophages treated with LPS, TNF-α mRNA and protein levels were lower in macrophages treated by LPS with azilsartan. In contrast, RANKL mRNA and protein expression levels in osteoblasts were the same in cells co-treated with azilsartan and LPS and those exposed to LPS only. Furthermore, azilsartan suppressed LPS-triggered MAPKs signaling pathway in macrophages. After 5-day supracalvarial injection, there is no difference between TNF-α injection group and TNF-α with azilsartan injection group. These findings imply that azilsartan prevents LPS-triggered TNF-α production in macrophages, which in turn prevents LPS-Triggered osteoclast formation and bone resorption in vivo.

Peonidin-3-O-glucoside and Resveratrol Increase the Viability of Cultured Human hFOB Osteoblasts and Alter the Expression of Genes Associated with Apoptosis, Osteoblast Differentiation and Osteoclastogenesis.

High-throughput RNA-sequencing can determine the impact of nutrients and their combinations on gene transcription levels in osteocytes, and clarify the biological pathways associated with their impact on bone tissues. Previously, we reported that resveratrol (RES) and peonidin-3-O-glucoside (POG) increased osteoblastogenesis, as well as reduced osteoclastogenesis in transgenic teleost fish models. Here, we perform whole-genome transcriptomic profiling of osteoblasts treated with POG or RES to provide a comprehensive understanding of alterations in gene expression and the molecular mechanisms involved. Cultured human fetal osteoblastic hFOB 1.19 cells were treated with the test compounds, and then RNA was used to prepare RNA-seq libraries, that were sequenced using a NovaSeq 6000. Treatment with POG or RES increased osteoblast proliferation and reduced apoptosis. Transcriptomic profiling showed that of the 29,762 genes investigated, 3177 were differentially expressed (1481 upregulated, 1696 downregulated, FDR ≤ 0.05) in POG-treated osteoblasts. In the RES-treated osteoblasts, 2288 genes were differentially expressed (DGEs, 1068 upregulated, 1220 downregulated, FDR ≤ 0.05). Ingenuity® Pathway Analysis (IPA) of DGEs from RES or POG-treated osteoblasts revealed significant downregulation of the apoptosis, osteoarthritis and HIF1α canonical pathways, and a significant reduction in Rankl mRNA expression. The data suggest that RES and POG have both anabolic and anticlastogenic effects.

Asiaticoside promotes heart autonomic nerve repair through receptor activator of nuclear factor kappa-B ligand pathway in coronary heart disease

This experiment intends to observe the effects of Asiaticoside on angiotensin II (AngII)-induced coronary heart disease (CHD) by regulating nuclear factor κ-B ligand receptor activator and RANK pathway (RANKL/RANK). Cardiomyocytes of rats with CHD were divided into AngII group (2 μmol/L), blank control group, and asiaticoside group (2 μmol/L and 10 μmol/L). After 24 hours of intervention, proliferation and apoptosis was assessed. At the same time, the expressions of RANKL, RANK and α-smooth muscle actin (α-SMA) were measured. Compared with blank group, the proliferation ability of cardiomyocytes with CHD in AngII group was significantly decreased and it increased after asiaticoside treatment (P <0.05). The apoptosis of cardiomyocytes in AngII group was higher than blank group and asiaticoside group. Cardiomyocytes proliferation in asiaticoside group was the most obvious (P <0.05). α-SMA expression decreased in AngII group and increased after asiaticoside treatment (P <0.05). RANKL and RANK mRNA in CHD were significantly upregulated after administration of asiaticoside (P <0.05). RANKL/RANK expression was high in normal CHD cardiomyocytes and reduced after Ang II stimulation. Asiaticoside promotes cardiomyocyte proliferation in CHD by promoting RANKL/RANK signaling, thereby reducing the possibility of heart failure in CHD.

Bone destruction in chronic otitis media is not mediated by the RANKL pathway or estrogen receptor-alpha.

Chronic otitis media with or without cholesteatoma progresses with various degrees of bone resorption and remodeling. Estrogen mediates osteoprotective effects through the receptor activator of NF-κB ligand (RANKL) pathway, which is mainly mediated by estrogen receptor-alpha (ER-α). The present study investigated the expression patterns of receptor activator of NF-κB (RANK), osteoprotegerin (OPG), RANKL, and ER-α in pathological tissue from patients with chronic otitis media to determine the roles of those factors in osteolytic mechanisms underlying the pathogenesis of chronic otitis media. Normal and pathological specimens from 18 patients with chronic otitis media were examined. There were no significant differences in RANK, OPG, RANKL, or ER-α mRNA expression between normal and pathological specimens of epithelial tissue. Our findings suggested that RANK, OPG, RANKL, and ER-α are not associated with the bone destruction in chronic otitis media; other cytokines may directly activate the osteoclasts in chronic otitis media.

Bone remodelling marker expression in grafted and ungrafted sheep tooth extraction sockets: A comparative randomised study

ObjectiveTo compare key markers of bone remodelling in a sheep tooth extraction model for sockets left to heal naturally or grafted with the bovine-derived xenograft Bio-Oss® covered with a collagen Bio-Gide® membrane. DesignRight side premolar teeth were removed from thirty Romney-cross ewes. Standardised sockets in each sheep were randomly allocated treatments, a grafted test and an empty control. At 4-, 8- and 16-weeks sheep were euthanized and tissue collected (N = 10/group). RANK, RANKL and OPG immunohistochemical analysis was performed (n = 3). RANK, RANKL, OPG, COL1A1, TIMP3, SP7 and MSX2 mRNA expression levels were determined using RT2-qPCR assays (n = 3). ResultsHistologically, more new woven bone was observed in the test group at all time points. Strong RANK and RANKL expression was found in both groups; at all time points with stronger RANK staining in the test group at 8 and 16 weeks. Strong OPG staining was localized to both osteoblasts and connective tissues. RANK receptor mRNA was expressed at a lower level in the test group (−4.26-fold; p = 0.02) at 4 weeks and SP7 at 16 weeks (−2.89-fold; p = 0.04). COL1A1 and TIMP3 mRNA expression increased significantly over time in the control group (p = 0.045, F = 5.4 and p = 0.003, F = 42.2 respectively). ConclusionSocket healing over time was comparable. The sheep tooth extraction model was found to be suitable for the evaluation of changes in the alveolar bone at the molecular level.

CXCR3 antagonist NBI-74330 mitigates joint inflammation in Collagen-Induced arthritis model in DBA/1J mice.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by uncontrolled synovial proliferation, pannus formation, cartilage injury, and bone destruction. We used the CXCR3-specific antagonist NBI-74330 to block T-cell-mediated signaling in a DBA/1J mouse model of collagen-induced arthritis (CIA). After CIA induction, DBA/1J mice were treated with NBI-74330 (100mg/kg) daily from day 21 until day 34 and evaluated for arthritic score and histopathological changes. Furthermore, using flow cytometry, we investigated the effects of NBI-74330 on Th1 (IFN-γ, TNF-α, T-bet, STAT4, Notch-3, and RANKL), Th17 (IL-21, IL-17A, STAT3, and RORγt), and Th22 (IL-22) cells in splenic CD4+ and CXCR3+T-cells. We also used RT-PCR to assess the effect of mRNA levels of IFN-γ, TNF-α, T-bet, RANKL, IL-17A, RORγt, and IL-22 in knee tissues. The IFN-γ, TNF-α, and IL-17A serum protein levels were measured using ELISA. Compared to vehicle-treated CIA mice, the severity of arthritic scores and histological severity of inflammation decreased significantly in NBI-74330-treated CIA mice. Moreover, compared to vehicle-treated CIA mice, the percentages of CD4+IFN-γ+, CD4+TNF-α+, CD4+T-bet+, CD4+STAT4+, CD4+Notch-3+, CXCR3+IFN-γ+, CXCR3+TNF-α+, CXCR3+T-bet+, CXCR3+STAT4+, CXCR3+Notch-3+, CD4+RANKL+, CD4+IL-21+, CD4+IL-17A+, CD4+STAT3+, CD4+RORγt+, and CD4+IL-22+ cells decreased in NBI-74330-treated CIA mice. Furthermore, NBI-74330-treatment downregulated IFN-γ, TNF-α, T-bet, RANKL, STAT3, IL-17A, RORγt, and IL-22 mRNA levels. Serum IFN-γ, TNF-α, and IL-17A levels were significantly lower in NBI-74330-treated CIA mice than in vehicle-treated CIA mice. This study demonstrates the antiarthritic effects of NBI-74330 in CIA mice. Therefore, these data suggest that NBI-74330 could be considered a potential RA treatment.

Changes in superoxide dismutase 3 (SOD3) expression in periodontal tissue during orthodontic tooth movement of rat molars and the effect of SOD3 on in vitro hypoxia-exposed rat periodontal ligament cells.

Hypoxia during orthodontic tooth movement (OTM) induces reactive oxygen species (ROS) production in periodontal tissues. Superoxide dismutase 3 (SOD3) is an anti-inflammatory enzyme that protects cells from ROS. This study investigated the expression and function of SOD3 during rat OTM and in hypoxia-exposed rat periodontal ligament (PDL) cells. OTM of right maxillary first molars were performed in 8-week-old male Sprague-Dawley rats using closed-coil spring for 1 and 14 days (n = 6 per group). SOD3 and hypoxia-inducible factor 1-alpha (HIF-1α) protein expression was evaluated by immunohistochemistry. The effects of SOD3 on cell viability and proliferation, ROS production, and mRNA expression of Hif1-α, receptor activator of nuclear factor kappa-Β ligand (Rankl), and osteoprotegerin (Opg) in PDL cells and osteoclast differentiation were investigated under normal and hypoxic conditions. SOD3 expression in PDL tissues significantly decreased on the compression side on day 1 and on both sides on day 14 of OTM. HIF-1α levels significantly increased on the compression side on day 14. Cell viability, cell proliferation, and Opg mRNA expression decreased, whereas ROS production and Hif1-α and Rankl mRNA expression increased in the PDL cells upon SOD3 silencing. Hypoxia reduced Sod3 and Opg mRNA expression and increased ROS, Rankl mRNA expression, and osteoclast formation; SOD3 treatment attenuated these effects. SOD3 plays a role in periodontal tissue remodelling during OTM and in hypoxia-exposed PDL cells through ROS, HIF-1α, and RANKL/OPG pathways. Moreover, SOD3 treatment could attenuate the negative effects of hypoxia on the PDL cells.

OR21-4 Comparison of the Effects of PTH (1-34), PTHrP (1-36) and Abaloparatide (ABL) on the Murine Osteoblast Transcriptome

Abstract Osteoporosis is a prevalent disease with substantial morbidity/mortality among the aging population. Due to gaps in knowledge, current therapeutics are limited in their ability to prevent degeneration of bone while also stimulating formation of new bone. Teriparatide (PTH (1-34)) and its analogs PTHrP (1-36) and abaloparatide (ABL), have been utilized for treatment of osteoporosis but have significant limitations in efficacy over long-term use. Research from our laboratory has shown PTH (1-34), PTHrP (1-36), and ABL exert time and dose-dependent differential responses in the osteoblast, leading us to hypothesize they may also differentially modulate the osteoblast transcriptome. In this study we show that treatment of mouse calvarial osteoblasts with 1 nM of these peptides for 4 h results in differing effects on the osteoblast transcriptome by performing gene enrichment analysis of RNA-Seq data. Genes were selected with a Log2 fold change >1 and a false discovery rate <0.05. These data were analyzed and compiled into heat maps for each peptide and smear/volcano plots. RNA-Sequencing revealed that PTH (1-34) regulated 367 genes, 194 were unique; PTHrP (1-36) regulated 117 genes, 15 were unique; ABL regulated 179 genes, 20 were unique. There were 74 genes shared only among PTH(1-34) and ABL; 16 genes shared only among PTH (1-34) and PTHrP; and 83 genes shared among all three peptides. Data collected show pathway-specific differences including, 1) cAMP/PKA, 2) Wnt/β-catenin, 3) Transcriptional regulation 4) Inflammatory response 5) Transmembrane transport, 6) Metabolism, and 7) NF-kB. Further analysis of the data illuminated that the three peptides increased Vitamin D receptor (VDR) and Cbp/p300-interacting transactivator 1 (CITED1) mRNAs similar to RankL expression. These findings were confirmed via qRT-PCR of additional cultured samples of mouse calvarial osteoblasts, treated with 1 nM of PTH (1-34), PTHrP (1-36), and ABL for 4 h prior to harvest. RankL mRNA, VDR mRNA, and CITED1 mRNA were measured for each sample and statistical differences were analyzed via Kruskal Wallis p<0.05. Additionally, we analyzed mRNA levels of several genes of interest, including WNT7b, WNT11, TCF7, SFRP4, FZD5, PP2R2A, and DVL3 mRNA. Pathway analysis and subsequent qRT-PCR confirmation has shown that PTH (1-34) and ABL lead to a significant increase in Wnt11 mRNA, while PTHrP (1-36) does not. Our findings highlight the complexity of the genetic and functional events triggered by PTH (1-34) and its analogs. Many studies have examined PTH signaling in the osteoblast/osteocyte; ours is the first to examine global effects of these peptides on the osteoblast transcriptome. Further delineation of which signaling events are attributable to PTH (1-34), PTHrP (1-36), and ABL exclusively and which are shared among all three will further our understanding of the effects these peptides have on the osteoblast and lead to refinement of PTH-derived treatments for osteoporosis. Presentation: Monday, June 13, 2022 11:45 a.m. - 12:00 p.m.

PSAT158 Dimeric [Cys25]PTH(1-34) and Monomeric PTH(1-34) Differentially Regulate the Expression of Genes Related to Calcium Homeostasis in Bone and Kidney

Abstract Osteoporosis and its resulting fractures threaten health and life of many patients worldwide; therefore, the development of effective and safe drugs for osteoporosis is an urgent need. In our previous report, dimeric [Cys25]PTH(1-34) peptide with arginine-to-cysteine mutation at position 25 (Dimer) showed a substantial osteo-anabolic effect similar or superior to that of PTH(1-34) in several mouse models. Surprisingly, cAMP was not elevated in serum or urine the Dimer-treated group unlike in the PTH(1-34)-treated group. From this, we hypothesized that the Dimer stimulates anabolic responses through a signaling pathway different from the cAMP-PKA pathway. To test this hypothesis, we assessed responses down-stream of the activated signal cascade from PTHR1 using GPCR Phospho Antibody Array in cells, as well as the expression levels of mRNAs and proteins related to calcium metabolism in bones and kidneys of mice. Cell lysates obtained from GP2.3 cells (treated with PTH(1-34) and Dimer with PBS as a control) were applied to the GPCR-MAPK Pathway Phosphorylation Antibody Array (Full Moon BioSystems, USA), containing 193 antibodies. Each of the antibodies has 6 replicates that are printed on standard-size coated glass microscope slides. The results of the Phospho-antibody array were further confirmed by Western Blot assay. Eighteen CD1 mice of 9 WO were divided into three groups: control, PTH(1-34)-treated, and Dimer-treated groups. 40 µg/kg of PTH(1-34) and 80 µg/kg of the Dimer were subcutaneously injected for each of the experimental groups, respectively. Then, two hours after the injection, the animals were sacrificed, and their kidneys and tibiae were harvested. Total mRNA samples from kidneys and tibiae were extracted using trizol reagent. Protein samples were extracted via protein extraction buffer. We estimated mRNA and protein levels of biomarkers related to calcium regulation via RT-PCR and ELISA with harvested bone and kidney tissues. Several changes of phosphorylation of signaling proteins in GP2.3 cells were detected between Dimer-treated group and PTH(1-34)-treated group. Expression of mRNA and protein levels of RANKL was lower in the Dimer-treated group compared to the PTH(1-34)-treated group. In contrast, expression of mRNA level of OPG in tibia samples was lower in the Dimer-treated group. In kidney, the mRNA and protein levels of 1α(OH)ase in the Dimer-treated group were lower than in the PTH(1-34)-treated group. However, mRNA level of 24(OH)ase was higher in the Dimer-treated group than in the PTH(1-34)-treated group. Other parameters were not significantly different between the Dimer-treated and PTH(1-34)-treated groups. We speculate that the osteoanabolic effect of Dimer uses a signaling pathway different from the cAMP-PKA pathway, regulating different expression parameters related to calcium metabolism in both the bone and kidney. Further studies are required to define exact signaling pathway that Dimer acts on. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

The Effects of Vitamin E Analogues α-Tocopherol and γ-Tocotrienol on the Human Osteocyte Response to Ultra-High Molecular Weight Polyethylene Wear Particles

Polyethylene (PE) liners are a common bearing surface of orthopaedic prostheses. Wear particles of ultra-high molecular weight PE (UHMWPE) contribute to periprosthetic osteolysis, a major cause of aseptic loosening. Vitamin E is added to some PE liners to prevent oxidative degradation. Osteocytes, an important cell type for controlling both bone mineralisation and bone resorption, have been shown to respond UHMWPE particles by upregulating pro-osteoclastogenic and osteocytic osteolysis. Here, we examined the effects of the vitamin E analogues α-tocopherol and γ-tocotrienol alone or in the context of UHMWPE particles on human osteocyte gene expression and mineralisation behaviour. Human osteoblasts differentiated to an osteocyte-like stage were exposed to UHMWPE wear particles in the presence or absence of either α-Tocopherol or γ-Tocotrienol. Both α-Tocopherol and γ-Tocotrienol induced antioxidant-related gene expression. UHMWPE particles independently upregulated antioxidant gene expression, suggesting an effect of wear particles on oxidative stress. Both vitamin E analogues strongly induced OPG mRNA expression and γ-Tocotrienol also inhibited RANKL mRNA expression, resulting in a significantly reduced RANKL:OPG mRNA ratio (p < 0.01) overall. UHMWPE particles reversed the suppressive effect of α-Tocopherol but not of γ-Tocotrienol on this pro-osteoclastogenic index. UHMWPE particles also upregulated osteocytic-osteolysis related gene expression. Vitamin E analogues alone or in combination with UHMWPE particles also resulted in upregulation of these genes. Consistent with this, both vitamin E analogues promoted calcium release from mineralised cultures of osteocyte-like cells. Our findings suggest that while vitamin E may suppress osteocyte support of osteoclastogenesis in the presence of UHMWPE particles, the antioxidant effect may induce osteocytic osteolysis, which could promote periprosthetic osteolysis. It will be important to conduct further studies of vitamin E to determine the long-term effects of its inclusion in prosthetic materials.

QingreHuoxue decoction protects joint and toe bone morphology and structure in rats with active RA through bidirectional regulation of bone metabolism

QingreHuoxue decoction (QRHX) is a well-known traditional Chinese medicine formula for treating rheumatoid arthritis (RA). It is not known how QRHX is involved in bone metabolism regulation and thereby alleviatesbone injury caused by arthritis. In this study, we used collagen-induced arthritis (CIA) rat model to evaluate the effects of QRHX onarthriticdiarthrodial joints and the underlying molecular mechanisms. Our results showed that QRHX significantly reduced foot joint volume, synovial inflammatory infiltration and the arthritis index (AI) score. Micro-CT imaging showed that toe bone and ankle joint were eroded and destroyed progressively during RA process, andcompensatory hyperplasia appeared in late stage RA. QRHX treatment inhibited foot bone erosion of early and mid-stage RA, and improved bone mineral density and trabecular fine structure in late RA.This studyunlocked the whole process details of ankle and toe bone injuryduring RA, as well as the bone metabolic characteristics under QRHX treatmentin CIArats. QRHX was confirmed to inhibite the expression of RANKL and DKK-1 mRNA and protein, enhance the expression of β-catenin. Meanwhile, QRHX reduced the pro-inflammatorycytokines IL-17, IL-1β and TNF-αand upregulated anti-inflammatory IL-4 in serum, improving local and systemic inflammatory microenvironment. These results demonstrated that QRHX had sustained protective effects on diarthrodial joints/bone during RA by a synergistic regulation mechanism, which reverses the imbalance of bone metabolismthroughinhibiting RANKL related osteoclast pathway and promotingWnt/β-catenin related osteoblast activation pathway.

Efficacy and Mechanisms of Oleuropein in Postmenopausal Osteoporosis.

Postmenopausal osteoporosis (PMOP) has a supernal morbidity rate in elderly females. To appraise the effects of oleuropein on bone densitometry, bone metabolic index, oxidative stress, and inflammatory index in PMOP. In addition, the mechanism of olive bittersweet preventing bone loss was explored. We grouped 80 salubrious female Sprague-Dawley rats into four teams: (1) sham operation team (sham, N = 20), (2) ovariectomy (OVX, N = 20), (3) castrated mice fed with oleuropein (OVX+ole, N = 20), and (4) castrated mice fed with estrogen (OVX+E2, N = 20). The ovariectomized SD rats were continuously raised with 200 μg/kg/dose of oleuropein. Bone mineral density and bone metabolism indexes were recorded. In order to assess the effectiveness of oleuropein on osteopenia, an enzyme-linked immunosorbent assay (ELISA) was devoted to examining the bone marrow indexes. The bone metabolism standards of PMOP rats were appraised by assessing serum levels of calcium, alkaline phosphatase (ALP), phosphorus, malondialdehyde (MDA), and nitrate content by experimental detection methods and levels of osteoclastogenesis inhibitory factor (OPG) and receptor activator for nuclear factor-κB ligand (RANKL) by ELISA. The OPG-RANK-RANKL signal passage was examined by Western blot (WB). We measured bone mineral density using dual-energy X-rays. Our animal experimental results indicated that oleuropein could significantly improve the bone mineral density of ovariectomized SD rats. In the meantime, it could reduce ending interleukin-6 (IL-6), malondialdehyde (MDA), nitrate, alkaline phosphatase (ALP), and phosphorus (P) serum concentration and would not affect Ca2+ concentration. In cell experiments, oleuropein also can promote the proliferation of osteoblasts. Furthermore, it can promote the expression of OPG protein and mRNA. In reverse, it inhibits the expression of RANKL protein and mRNA. Oleuropein can not only improve the inflammatory and oxidative indexes of castrated rats but also prevent osteoporosis. Oleuropein avoids bone resorption by regulating OPG/RANKL expression.

RESPONSE OF THE PERIODONTAL TISSUES TO β-ADRENERGIC STIMULATION

Objectives Stress is a risk factor for periodontitis, and β-adrenergic receptor (βAR) stimulation increases angiogenesis on long bones. Enhanced periodontal vascularization could act as a potential host susceptibility factor. The aim was to investigate the mandibular angiogenic response to βAR stimulation. Study Design Twenty C57BL6 female mice received daily intraperitoneal injections of isoproterenol (ISO) or phosphate-buffered saline (PBS) for 6 weeks. Hemimandibles and tibias were collected for immunolocalization of endomucin, tyrosine hydroxylase, neuropeptide Y, and norepinephrine transporter (NET). Vascular Endothelial Growth Factor A, IL-1β, IL-6, Adrb2, and Rankl mRNA expression were assessed 2 hours after PBS or ISO treatment. Results Despite similar sympathetic innervation and Adrb2 expression in mandibular tissues and tibias, ISO treatment did not increase endomucin+ vessel area or the total number of endomucin+ vessels in the mandible. Moreover, the expression of VEGFa, IL-6, IL-1β, and Rankl in the molar region did not change following ISO administration. We also detected high expression of NET by immunofluorescence in mandibular alveolar osteoblasts, osteocytes, and periodontal ligament fibroblasts, which was confirmed by quantitative polymerase chain reaction, compared to the tibia from the same animals. Conclusions Stress and sympathetic nervous system activation increase the risk of periodontitis via mechanisms other than change in microvascular density and possibly requiring inflammatory cells. Stress is a risk factor for periodontitis, and β-adrenergic receptor (βAR) stimulation increases angiogenesis on long bones. Enhanced periodontal vascularization could act as a potential host susceptibility factor. The aim was to investigate the mandibular angiogenic response to βAR stimulation. Twenty C57BL6 female mice received daily intraperitoneal injections of isoproterenol (ISO) or phosphate-buffered saline (PBS) for 6 weeks. Hemimandibles and tibias were collected for immunolocalization of endomucin, tyrosine hydroxylase, neuropeptide Y, and norepinephrine transporter (NET). Vascular Endothelial Growth Factor A, IL-1β, IL-6, Adrb2, and Rankl mRNA expression were assessed 2 hours after PBS or ISO treatment. Despite similar sympathetic innervation and Adrb2 expression in mandibular tissues and tibias, ISO treatment did not increase endomucin+ vessel area or the total number of endomucin+ vessels in the mandible. Moreover, the expression of VEGFa, IL-6, IL-1β, and Rankl in the molar region did not change following ISO administration. We also detected high expression of NET by immunofluorescence in mandibular alveolar osteoblasts, osteocytes, and periodontal ligament fibroblasts, which was confirmed by quantitative polymerase chain reaction, compared to the tibia from the same animals. Stress and sympathetic nervous system activation increase the risk of periodontitis via mechanisms other than change in microvascular density and possibly requiring inflammatory cells.