mRNA Levels Of OPG Research Articles

Abnormal amyloid precursor protein processing in periodontal tissue in a murine model of periodontitis induced by Porphyromonas gingivalis.

The study aimed to investigate the change of amyloid precursor protein (APP) processing and amyloid β (Aβ) metabolites in linking periodontitis to Alzheimer's disease (AD). Aβ is one of the main pathological features of AD, and few studies have discussed changes in its expression in peripheral tissues or analyzed the relationship between the peripheral imbalance of Aβ production and clearance. A murine model of periodontitis was established by oral infection with Porphyromonas gingivalis (P. gingivalis). Micro-computed tomography (Micro-CT) was used to observe the destruction of the alveolar bone. Nested quantitative polymerase chain reaction (qPCR) was used to measure small quantities of P.gingivalis DNA in different tissues. Behavioral experiments were performed to measure cognitive function in the mice. The mRNA levels of TNF-α, IL-6, IL-8, RANKL, OPG, APP695, APP751, APP770, and BACE1 in the gingival tissues or cortex were detected by RT-PCR. The levels of Aβ1-40 and Aβ1-42 in gingival crevicular fluid (GCF) and plasma were tested by ELISA. P. gingivalis oral infection was found to cause alveolar bone resorption and impaired learning and memory. P.gingivalis DNA was detected in the gingiva, blood and cortex of the P.gingivalis group by nested qPCR (p < .05). The mRNA expression of TNF-α, IL-6, IL-8, RANKL/OPG, and BACE1 in the gingival tissue was significantly higher than that in the control group (p < .05). Similarly, upregulated mRNA levels of APP695 and APP770 were observed in the gingival tissuses and cortex of the P. gingivalis group (p < .05). The levels of Aβ1-40 and Aβ1-42 in the GCF and plasma of the P. gingivalis group were significantly higher than those in the control group (p < .05). P. gingivalis can directly invade the brain via hematogenous infection. The invasion of P. gingivalis could trigger an immune response and lead to an imbalance between Aβ production and clearance in peripheral tissues, which may trigger an abnormal Aβ metabolite in the brain, resulting in the occurrence and development of AD.

The direct impact of pegvisomant on osteoblast functions and bone development.

Acromegaly is a chronic disease characterized by growth hormone (GH) hypersecretion, usually caused by a pituitary adenoma, resulting in elevated circulating levels of insulin-like growth factor type I (IGF-I). Pegvisomant (PEG), the GH-receptor (GHR) antagonist, is used in treating acromegaly to normalize IGF-I hypersecretion. Exposure to increased levels of GH and IGF-I can cause profound alterations in bone structure that are not completely reverted by treatment of GH hypersecretion. Indeed, there is evidence that drugs used for the treatment of acromegaly might induce direct effects on skeletal health regardless of biochemical control of acromegaly. We investigated, for the first time, the effect of PEG on cell proliferation, differentiation, and mineralization in the osteoblast cell lines MC3T3-E1 and hFOB 1.19 and its potential impact on bone development in zebrafish larvae. We observed that PEG did not affect osteoblast proliferation, apoptosis, alkaline phosphatase (ALP) activity, and mineralization. After PEG treatment, the analysis of genes related to osteoblast differentiation showed no difference in Alp, Runx2, or Opg mRNA levels in MC3T3-E1 cells. GH significantly decreased cell apoptosis (-30 ± 11%, p < 0.001) and increased STAT3 phosphorylation; these effects were suppressed by the addition of PEG in MC3T3-E1 cells. GH and PEG did not affect Igf-I, Igfbp2, and Igfbp4 mRNA levels in MC3T3-E1 cells. Finally, PEG did not affect bone development in zebrafish larvae at 5days post-fertilization. This study provides a first evidence of the impact of PEG on osteoblast functions both in vitro and in vivo. These findings may have clinically relevant implications for the management of skeletal health in subjects with acromegaly.

Attenuative effects of collagen peptide from milkfish (Chanoschanos) scales on ovariectomy-induced osteoporosis.

Osteoporosis is characterized by low bone mass, bone microarchitecture disruption, and collagen loss, leading to increased fracture risk. In the current study, collagen peptides were extracted from milkfish scales (MS) to develop potential therapeutic candidates for osteoporosis. MS was used to synthesize a crude extract of fish scales (FS), collagen liquid (COL), and hydroxyapatite powder (HA). COL samples were further categorized according to the peptide size of total COL (0.1 mg/mL), COL < 1 kDa (0.1 mg/mL), COL: 1-10 kDa (0.1 mg/mL), and COL > 10 kDa (0.1 mg/mL) to determine it. Semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR) and immunofluorescence labeling were used to assess the expression levels of specific mRNA and proteins in vitro. For in vivo studies, mice ovariectomy (OVX)-induced postmenopausal osteoporosis were developed, while the sham surgery (Sham) group was treated as a control. Collagen peptides (CP) from MS inhibited osteoclast differentiation in RAW264.7 cells following an insult with nuclear factor kappa-B ligand (RANKL). CP also enhanced osteoblast proliferation in MG-63 cells, possibly through downregulating NFATc1 and TRAP mRNA expression and upregulating ALP and OPG mRNA levels. Furthermore, COL1 kDa also inhibited bone density loss in osteoporotic mice. Taken together, CP may reduce RANKL-induced osteoclast activity while promoting osteoblast synthesis, and therefore may act as a potential therapeutic agent for the prevention and control of osteoporosis.

Strontium chloride improves bone mass by affecting the gut microbiota in young male rats.

Bone mass accumulated in early adulthood is an important determinant of bone mass throughout the lifespan, and inadequate bone deposition may lead to associated skeletal diseases. Recent studies suggest that gut bacteria may be potential factors in boosting bone mass. Strontium (Sr) as a key bioactive element has been shown to improve bone quality, but the precise way that maintains the equilibrium of the gut microbiome and bone health is still not well understood. We explored the capacity of SrCl2 solutions of varying concentrations (0, 100, 200 and 400 mg/kg BW) on bone quality in 7-week-old male Wistar rats and attempted to elucidate the mechanism through gut microbes. The results showed that in a Wistar rat model under normal growth conditions, serum Ca levels increased after Sr-treatment and showed a dose-dependent increase with Sr concentration. Three-point mechanics and Micro-CT results showed that Sr exposure enhanced bone biomechanical properties and improved bone microarchitecture. In addition, the osteoblast gene markers BMP, BGP, RUNX2, OPG and ALP mRNA levels were significantly increased to varying degrees after Sr treatment, and the osteoclast markers RANKL and TRAP were accompanied by varying degrees of reduction. These experimental results show that Sr improves bones from multiple angles. Further investigation of the microbial population revealed that the composition of the gut microbiome was changed due to Sr, with the abundance of 6 of the bacteria showing a different dose dependence with Sr concentration than the control group. To investigate whether alterations in bacterial flora were responsible for the effects of Sr on bone remodeling, a further pearson correlation analysis was done, 4 types of bacteria (Ruminococcaceae_UCG-014, Lachnospiraceae_NK4A136_group, Alistipes and Weissella) were deduced to be the primary contributors to Sr-relieved bone loss. Of these, we focused our analysis on the most firmly associated Ruminococcaceae_UCG-014. To summarize, our current research explores changes in bone mass following Sr intervention in young individuals, and the connection between Sr-altered intestinal flora and potentially beneficial bacteria in the attenuation of bone loss. These discoveries underscore the importance of the "gut-bone" axis, contributing to an understanding of how Sr affects bone quality, and providing a fresh idea for bone mass accumulation in young individuals and thereby preventing disease due to acquired bone mass deficiency.

Acteoside Derived from Cistanche Improves Glucocorticoid-Induced Osteoporosis by Activating PI3K/AKT/mTOR Pathway

Objective Glucocorticoids are widely used in clinical practice; however, they can cause side effects, such as osteoporosis. Acteoside (ACT) from Cistanche has been used to combat a variety of diseases. The study was conducted to evaluate the efficacy of ACT in glucocorticoid-induced osteoporosis (GIOP) and its potential mechanism. Methods Dexamethasone (Dex) was injected intramuscularly to induce osteoporosis in a rat model, and ACT was given orally. ACT was supplemented in vivo in Dex-stimulated osteoblastic MC3T3-E1 cells. RT-qPCR was performed to assess the mRNA levels of bone formation (Runx2, CoL1A1), and bone resorption (OPG and RANKL). A commercial ELISA kit was applied to assess serum OC and CTX levels. Western blot was performed to assess protein levels in the PI3K/AKT/mTOR signaling pathway. CCK-8 assay and flow cytometry were performed to assess osteoblast viability and apoptosis. Results ACT reduced Dex-induced bone microstructure deterioration, increased serum levels of OC, and decreased the levels of CTX (P < 0.05). In the MC3T3-E1 cells, Dex inhibited cell viability and promoted apoptosis; however, this effect was greatly attenuated by ACT (P < 0.05). Concurrently, ACT reversed the reduction in Runx2, osterix, CoL1A1, and OPG mRNA levels, ALP activity, and the promotion of RANKL by Dex. Additionally, ACT attenuated Dex-induced inhibition of p-AKT/AKT, p-mTOR/mTOR, and p-PI3K/PI3K protein levels by Dex (P < 0.05), while the PI3K/AKT/mTOR pathway inhibitor LY294002 diminished the potential effect of ACT (P < 0.05). Conclusion ACT from Cistanche may exert osteoprotective effects by activating the PI3K/AKT/mTOR signaling pathway to alleviate Dex-induced osteoporosis.

The expression levels of MicroRNA-146a, RANKL and OPG after non-surgical periodontal treatment

ObjectiveMicroRNA-146a (miR-146a) is a regulator of inflammatory response. Periodontitis is a disease with immune pathophysiology of the periodontium in which the inflammation results in the destruction of the soft tissues and alveolar bone. Therefore, the aim of this study was to investigate the expressions of miR-146a, OPG, and RANKL in diseased and healthy periodontal tissues to understand whether miR-146a expression level may associate with OPG and RANKL mRNA levels and OPG/RANKL ratio after non-surgical periodontal treatment.MethodsThe levels of miR-146a, RANKL, and OPG in gingival tissues from patients with generalized periodontitis stages II and III and grades A and B (n = 15, group A), patients with generalized periodontitis stages III and IV and grade C (n = 15, group B), and healthy individuals (n = 10) were determined by real-time PCR. The associations of miR-146a expression with OPG and RANKL levels were evaluated.ResultsThe levels of miR-146a in two subgroups within periodontitis patients were significantly higher than healthy subjects (P < 0.0001). MiR-146a showed the increased level in group A of patients compared with group B (P < 0.05). Clinical parameters such as probing depth (PD) and clinical attachment loss (CAL) were significantly higher in patients than control group (P < 0.05). The levels of OPG and RANKL were increased in patients compared with healthy subjects, although the elevated levels were not statistically significant. MiR-146a was not associated with OPG and RANKL levels and OPG/RANKL ratio.ConclusionsThe results of this study failed to show the associations of miR-146a level with OPG and RANKL levels and OPG/RANKL ratio in periodontitis after non-surgical periodontal treatment.

The Variability in Cytocompatibility and Bone Conduction Based on Different Pore Size and Porosity of n-HA/PA66 Composite Scaffolds

As porous materials, nano-hydroxyapatite/polyamide 66 (n-HA/PA66) composite scaffolds with both desirable bioactivity and good mechanical properties showed great potential to reconstruct the bone defect. Moreover, the pore size and porosity played a key role in the scaffold architecture and cell or bone ingrowth. To investigate the cytocompatibility of different pore size and porosity of n-HA/PA66 composite scaffolds on differentiation and cytocompatibility of osteogenically induced bone marrow-derived mesenchymal stem cells (BMSCs) and bone conduction in repairing the calvarial critical size defect of Sprague-Dawley rats in vivo, we evaluated three kinds of n-HA/PA66 composite scaffolds according to the pore size and porosity in this study (group A: mean pore size was 214 ± 107.3 μm, and more than 70% were arranged in 100–300 μm; group B: material mean pore size was 375 ± 132.2 μm, and about 60% were distributed in 300–500 μm; group C: mean pore size was 533 ± 169.4 μm, and more than 60% were in 400–700 μm). Osteogenically induced BMSCs were seeded in the three types of n-HA/PA66 material and cultured in vitro, and the variability on cell adhesion, growth, proliferation, osteogenic differentiation was analyzed using scanning electron microscopy alkaline phosphatase (ALP) and collagen type I (COL I) immunohistochemical staining, as well as quantitative real-time PCR (qRT-PCR) analysis on the osteogenesis-related gene expression (alkaline phosphatase, COL I), was done. Three group matrices/BMSC composites were implanted into the cranial defect of Sprague-Dawley rats. The differentiations of osteogenesis in vivo were then evaluated by histological and qRT-PCR analysis on mRNA levels of OPG and RANKL after 4 and 8 weeks, respectively. The in vitro and in vivo results showed that the group B n-HA/PA66 scaffold was more suitable for osteogenically induced BMSC proliferation, differentiation in vitro, and bone conduction in vivo than groups A and C, indicating that the porous n-HA/PA66 matrices with a mean pore size of 375 ± 132.2 μm and porosity 77 ± 2.9% have better cell biocompatibility and bone conduction.

Leptin regulates OPG and RANKL expression in Gingival Fibroblasts and Tissues of Chronic Periodontitis Patients.

Objective: Chronic periodontitis is a bone-destructive disease affecting periodontal support structures. Although leptin has a protective effect against periodontitis, the underlying mechanism remains unclear. Therefore, this study aimed to investigate the possible role of leptin by examining its relationship with OPG and RANKL in human gingival tissues obtained from patients with chronic periodontitis.Method: Twenty-two patients with chronic periodontitis were enrolled (10 with moderate periodontitis and 12 with severe periodontitis) in the experimental group, and 12 healthy individuals were enrolled in the control group. Gingival tissue samples were collected, and the protein levels and localization of leptin, OPG, and RANKL were studied using immunohistochemistry (IHC). The staining intensities of leptin, OPG, and RANKL were correlated with the periodontal clinical index. Moreover, real-time quantitative PCR (RT-qPCR) was used to determine OPG and RANKL mRNA levels in gingival fibroblasts stimulated with gradient concentrations of leptin protein in vitro.Result: Leptin, OPG, and RANKL were located in the cytoplasm of gingival epithelial cells and the connective tissue. Leptin was widely and significantly expressed in the control group, whereas it was lightly stained in the severe group. RANKL was lightly stained in the control group, whereas it was widely and significantly expressed in the severe group. The control and the moderate groups had similar OPG levels, which were significantly higher than that in the severe group. Leptin was positively correlated with OPG(r = 0.905, p < 0.01) and negatively correlated with RANKL (r = -0.635, p < 0.01). In vitro low concentrations of leptin led to an increased OPG/RANKL mRNA ratio, whereas the opposite effect was observed at high concentrations.Conclusion: Leptin can regulate OPG and RANKL expression in gingival fibroblasts and may thus play a role in the development of chronic periodontitis by modulating the OPG/RANKL ratio.

Liraglutide Promotes Osteoblastic Differentiation in MC3T3-E1 Cells by ERK5 Pathway.

Liraglutide is a glucagon-like peptide-1 analogue widely used in the treatment of type 2 diabetes mellitus. However, the effects of liraglutide on osteoblast proliferation and differentiation in MC3T3-E1 cells have not been fully elucidated. In the present study, the promoting effects of liraglutide were investigated in MC3T3-E1 cells. The results indicated that cell viability was affected following the treatment of the cells with different concentrations of liraglutide (0, 10, 100, and 1000 nM) at different time periods of culture (24, 48, and 72 h). Moreover, the activity levels of alkaline phosphatase and the number of mineralized nodules in MC3T3-E1 cells were significantly increased following treatment with 100 nM liraglutide. The mRNA and protein levels of Col-1, OPG, and OCN in MC3T3-E1 cells were also markedly increased following 100 nM liraglutide treatment compared with those of the control group. The expression levels of the ERK5 signaling pathway key proteins (MEK5, p-ERK5, ERK5, and NUR77) were increased following liraglutide treatment in MC3T3-E1 cells, and the gene expression levels of the ERK5 signaling pathway were also elevated. Moreover, the ERK5 inhibitor XMD8-92 significantly decreased the expression levels of p-ERK5 and NUR77 as well as the proliferation of osteoblasts. However, these changes could be rescued by liraglutide to some extent. Therefore, these results revealed that liraglutide may promote osteoblastic differentiation and proliferation in MC3T3-E1 cells via the activation of the ERK5 signaling pathway.

Suppressing PLCγ1 enhances osteogenic and chondrogenic potential of BMSCs

Phosphatidylcholine-specific phospholipase Cγ1 (PLCγ1) is involved in regulating cell metabolism. However, little is known how PLCγ1 directs BMSC differentiation. Here, we investigated the role of PLCγ1 in rat BMSC differentiation into osteoblasts and chondrocytes. The results of Alizarin red and Alcian blue staining showed that PLCγ1 inhibitor U73122 significantly enhanced the mineralization capacity and proteoglycan deposition of BMSCs. The results of qPCR technique and Western blot analysis showed that long-term treatment of U73122 enhanced COL1A1 and OPG mRNA levels and Collagen 1A1, BMP2, and p-Smad1/5/9 protein levels and that short-term treatment of U73122 enhanced COL2A1 and SOX9 mRNA levels and Collagen 2, SOX9, Aggrecan, TGF-β3, and p-Smad2/3 protein levels. Decreased p-mTOR and p-P38 contributed to enhanced osteogenic potentials of BMSCs and increased p-P38 contributed to enhanced chondrogenic potentials of BMSCs. The scaffold transplantation with U73122+BMSC was more efficacious than BMSC alone for osteochondral defect repair in a rat model. Therefore, suppressing PLCγ1 could improve the capacity to effectively use BMSCs for cell therapy of osteochondral defect.

Remodeling process in bone of aged rats in response to resistance training

AimsWe investigate the effects of RT on the mechanical function, gene, and protein expression of key factors involved in bone remodeling during aging. Main methodsMale rats of 3 and 21 months of age were randomly allocated into four groups (8 per group): young sedentary (YS), young trained (YT), old sedentary (OS), and old trained (OT). RT was performed three times per week (12 weeks). Bone tenacity and stiffness were measured by biomechanical tests and mRNA levels of COL1A1, MEPE, SOST, OPG, BMP-2, PPAR-y, MMP-2-9-13, and TIMP-1 were evaluated by quantitative PCR. COL1A1 protein and MMP-2 activity were detected by western blotting and zymography assays. Key findingsAging increased stiffness, while BMP-2, OPG, COL1A1 and MMP-2 mRNA levels reduced (OS vs YS; p ≤ 0.05). RT increased the tenacity of the femur and reduced PPAR-γ regardless of age (YT vs. YS; OT vs. OS; p ≤ 0.05). RT downregulated SOST mRNA levels only in the OT group (vs. OS group, p ≤ 0.05). RT mitigated the age-associated increase in MMP-9 mRNA levels (p ≤ 0.05). In young animals, upregulation in MEPE, MMP-13, TIMP-1 were observed after RT, as well an increase in COL1A1 protein and MMP-2 activity (p ≤ 0.05). SignificanceRT improved bone tenacity independent of aging, which is relevant for mechanical function, while, at protein levels, RT upregulated MMP-2 activity and collagen 1 only in young rats. This study highlights the importance of exercise on bone health and identifies specific molecular changes in response to RT. Our findings provide insights into the mechanisms involved in age-related changes.

S-amlodipine improves endothelial dysfunction via the RANK/RANKL/OPG system by regulating microRNA-155 in hypertension

S-amlodipine has been broadly used to treat hypertension, but its protective effects and underlying mechanism remain controversial. The purpose of our study was to investigate the mechanism by which S-amlodipine improves endothelial dysfunction. Specifically, we investigated if S-amlodipine regulates RANK/RANKL/OPG and micro-RNA 155 (miR-155) levels. Spontaneous hypertensive rats (SHR) were randomly divided into two groups: SHR (n = 12) and S-amlodipine (n = 12). We found that left ventricular ejection fraction (LVEF) increased significantly in the S-amlodipine group compared to the SHR group. After 10 weeks of S-amlodipine treatment, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were significantly lower and eNOS and NO production was significantly higher in the S-amlodipine group compared to the SHR group. In human umbilical vein endothelial cells (HUVECs), miR-155, RANK, and RANKL levels were significantly decreased, while OPG mRNA levels were significantly increased in the S-amlodipine group. HUVECs were transfected with miR-155 mimics or an inhibitor to determine the relationship between miR-155 and RANK/RANKL/OPG and NF-κB signaling. OPG mRNA levels following miR-155 inhibition were significantly higher compared to levels following treatment with miR-155 mimics. S-amlodipine significantly inhibited RANKL expression and NF-κB phosphorylation, and there were no significant differences in response to the NF-κB inhibitor (Bay110785). RANKL expression and NF-κB phosphorylation significantly decreased in the miR-155 inhibitor group. Furthermore, OPG protein expression significantly increased in response to miR-155 inhibition and S-amlodipine treatment (all p < 0.05). Our results indicate that S-amlodipine inhibits inflammation and protects against endothelial dysfunction, likely via regulating the RANK/RANKL/OPG pathway, which appears to be downstream of miR-155.

Implication of endothelin-1 in human aortic valve calcification

Aortic Stenosis (AS) is the most common acquired valvulopathy in the Western world. AS is characterized by the differenciation of valvular interstitial cells (VICs) into osteoblastic cells, leading to valvular calcification. The physiopathology of AS is not fully understood and there is, to date, no treatments to stop or slow the progression of AS. The purpose of our study was to investigate the role of endothelin-1 (ET-1) on VICs calcification. Primary human valvular interstitial cells (hVICs) were first cultured in pro-osteogenic medium or incubated with ET-1. hVICs were also co-cultured with human valvular endothelial cells (hVECs) obtained from patients with (pathological) or without (healthy) AS in Transwell™ inserts. hVICs calcification was quantified using o-cresolphthalein assay. Real-time quantitative PCR (qRT-PCR) and Western blot analysis were performed to determine the expression of ET-1 (EDN1), inflammation (IL1B, IL6) and bone markers (RUNX2, OPG, OPN). Immunochemistry was performed using ACTA2 antibody to evaluate the phenotype of VICs. hVICs showed an increase of calcification and a decrease of ACTA2 when cultured in pro-osteogenic medium or co-cultured with pathological hVECs. qRT-PCR revealed higher mRNA levels of EDN1, RUNX2, OPG, OPN, IL1B and IL6. At the same time, fibroblast marker ACTA2 was decreased. VIC fibroblastic phenotype was restored and calcification decreased when hVICs were co-cultured with healthy hVECs or incubated with ET-1 antagonist. The present study suggests that ET-1 play a role in the pathogenesis of AS, and therefore, pharmacological ET-1 antagonists could be used to attenuate or stop VIC calcification.

A novel effect of geraniin on OPG/RANKL signaling in osteoblasts

In this study, the effects of geraniin on osteoprotegerin/receptor activator of nuclear factor-κB ligand(OPG/RANKL) in regulating the proliferation of osteoblasts and suppression of osteoclast-like cells (OLC) in OLC-osteoblast co-cultured system in vitro were investigated. Osteoblasts were cultured and identified with alkaline phosphatase (ALP), gomori stain, and mineralized nodule stain. OLCs were isolated from long bones of Sprague-Dawley (SD) rats and identified with tartrate-resistant acid phosphatase(TRAP) stain. Methyl thiazolyl tetrazolium assay was used to examine the proliferation of osteoblasts, and immunocytochemistry and in situ hybridization to analyze the expression OPG/RANKL in osteoblasts co-cultured with osteoclasts under the action of geraniin, respectively. Geraniin could regulate the proliferation of osteoblasts MC3T3-E1, decrease the number of OLC in OLC-osteoblast co-cultured system, and inhibit the bone resorption areas and resorption pits of OLC in vitro experiments. Geraniin could promote the mRNA and protein expression levels of OPG and suppress those of RANKL in osteoblasts. These results indicate that geraniin has a promoting effect on the proliferation of osteoblasts and an inhibitory effect on the osteoclastic bone-resorption through regulating OPG/RANKL signaling pathway in OLC-OB co-cultured system.

Mechanism of genistein regulating the differentiation of vascular smooth muscle cells into osteoblasts via the OPG/RANKL pathway.

ObjectiveThe present study aimed to investigate the mechanism of genistein, a tyrosine kinase inhibitor, regulating the differentiation of vascular smooth muscle cells (VSMCs) into osteoblasts via the OPG/RANKL (Osteoprotegerin/Receptor Activator of Nuclear Factor-κB Ligand) pathway.MethodsThe mouse VSMCs were isolated, purified and cultured. We constructed the LV5-Tnfrsf11b overexpression lentiviral vector and LV3-OPG-309 interference lentiviral vector. The OPG overexpression was induced and the growth of VSMCs infected with the lentiviral vector was observed. The VSMC calcification and control group were treated with different doses of genistein. The mRNA and protein expression levels of OPG, α-SM-actin (smooth muscle actin), ALP (alkaline phosphatase) and OPN (osteopontin) were detected in VSMCs after treatment using RT-PCR and Western Blot.ResultWe induced OPG overexpression and performed lentiviral vector infection of the VSMCs to suppress OPG expression, respectively, which was followed by treatment with genistein. The results showed that the relative expression of OPG was the highest in the VSMC calcification +genistein +OPG overexpression-inducing treatment group. It was the lowest in the VSMC calcification +OPG expression-suppressing treatment group. The relative expression of ALP was the highest in the VSMC calcification +OPG expression-suppressing treatment group, and the lowest in the VSMCs+genistein treatment group.ConclusionOPG gene plays an important regulatory role in the growth of VSMCs, by suppressing the calcification of VSMCs. Genistein could regulate the differentiation of VSMCs into osteoblasts via the OPG/RANKL pathway in a dose-dependent manner.

Wnt/β-catenin signaling plays an important role in the protective effects of FDP-Sr against oxidative stress induced apoptosis in MC3T3-E1 cell

Strontium fructose 1,6-diphosphate (FDP-Sr) is a new strontium-containing compound. The primary aim of this study was to clarify whether the structure component of FDP-Sr, FDP could benefit the protective effect of Sr (II) against oxidative stress induced apoptosis, and meanwhile to further explore the important role of Wnt/β-catenin signaling in the anti-apoptosis effect of FDP-Sr in response to oxidative stress induced by H2O2 in an osteoblastic MC3T3-E1 cell line. Results showed that FDP-Sr could improve the osteoblastic differentiation under oxidative stress with induced cell proliferation and improved mineralization. The inhibition effect of FDP-Sr on cell apoptosis induced by H2O2 was proved by reduced reactive oxygen species production and activated caspase3. Under oxidative stress, mRNA and protein levels of phospho-β-catenin reduced, while β-catenin increased in the FDP-Sr treatment cell, leaded to the up-regulations of Runx2 and OPG at both mRNA and protein levels, finally improved the differentiation of osteoblasts. By the engagement of Wnt/β-catenin pathway’s inhibitor (XAV-939), the protective effects of FDP-Sr on osteoblastic differentiation against oxidative stress were repressed along with inhibited wnt/β-catenin signaling and reduced mRNA and protein levels of Runx2 and OPG. In conclusion, FDP-Sr was demonstrated to protect osteoblast differentiation from oxidative damage induced by H2O2 through up-regulation of Wnt/β-catenin signaling, and FDP in FDP-Sr was able to directly improve the oxidative stress injury through its ROS scavenging ability.

MicroRNA-145 Mediates Steroid-Induced Necrosis of the Femoral Head by Targeting the OPG/RANK/RANKL Signaling Pathway.

ObjectiveTo investigate the role of microRNA-145 (miR-145) in steroid-induced necrosis of the femoral head (SINFH) by evaluating its effects on the OPG/RANK/RANKL signaling pathway.MethodsA rat model of SINFH was constructed via injection of the lentiviral vector pLV-shRNA-miR-145. Pathological observation was performed via tartrate-resistant acid phosphatase (TRAP) staining, and serum OPG levels were detected by ELISA. The mRNA expression levels of miR-145, OPG, RANK and RANKL in THP-1 cells were assessed by RT-PCR, and the protein expression levels of OPG, RANK and RANKL were assessed by western blotting.ResultsThe expression of miR-145 in the lentivirus-mediated miR-145 group was significantly up-regulated compared with that in the control and normal groups (both P < 0.01). Serum OPG levels were decreased in SINFH rats compared with control and normal rats. The mRNA and protein expression levels of OPG in THP-1 cells decreased after transfection (all P < 0.05). By contrast, the mRNA and protein expression levels of RANK and RANKL in THP-1 cells increased after transfection (all P < 0.05). After transfection of 293T cells with an miR-145 overexpression vector, miR-145 expression in 293T cells increased significantly, while OPG mRNA and protein expression decreased significantly (all P < 0.05).ConclusionMiR-145 plays a role in the occurrence of SINFH by targeting the OPG/RANK/RANKL signaling pathway.

Targeted disruption of BMP signaling through type IA receptor (BMPR1A) in osteocyte suppresses SOST and RANKL, leading to dramatic increase in bone mass, bone mineral density and mechanical strength

Recent studies suggest a critical role of osteocytes in controlling skeletal development and bone remodeling although the molecular mechanism is largely unknown. This study investigated BMP signaling in osteocytes by disrupting Bmpr1a under the Dmp1-promoter. The conditional knockout (cKO) mice displayed a striking osteosclerotic phenotype with increased trabecular bone volume, thickness, number, and mineral density as assessed by X-ray and micro-CT. The bone histomorphometry, H&E, and TRAP staining revealed a dramatic increase in trabecular and cortical bone masses but a sharp reduction in osteoclast number. Moreover, there was an increase in BrdU positive osteocytes (2–5-fold) and osteoid volume (~4-fold) but a decrease in the bone formation rate (~85%) in the cKO bones, indicating a defective mineralization. The SEM analysis revealed poorly formed osteocytes: a sharp increase in cell numbers, a great reduction in cell dendrites, and a remarkable change in the cell distribution pattern. Molecular studies demonstrated a significant decrease in the Sost mRNA levels in bone (>95%), and the SOST protein levels in serum (~85%) and bone matrices. There was a significant increase in the β-catenin (>3-fold) mRNA levels as well as its target genes Tcf1 (>6-fold) and Tcf3 (~2-fold) in the cKO bones. We also showed a significant decrease in the RANKL levels of serum proteins (~65%) and bone mRNA (~57%), and a significant increase in the Opg mRNA levels (>20-fold) together with a significant reduction in the Rankl/Opg ratio (>95%), which are responsible for a sharp reduction in the cKO osteoclasts. The values of mechanical strength were higher in cKO femora (i.e. max force, displacement, and work failure). These results suggest that loss of BMP signaling specifically in osteocytes dramatically increases bone mass presumably through simultaneous inhibition of RANKL and SOST, leading to osteoclast inhibition and Wnt activation together. Finally, a working hypothesis is proposed to explain how BMPR1A controls bone remodeling by inhibiting cell proliferation and stimulating differentiation. It is reported that RANKL and SOST are abundantly expressed by osteocytes. Thus, BMP signaling through BMPR1A plays important roles in osteocytes.

Serum from patients with ankylosing spondylitis can increase PPARD, fra-1, MMP7, OPG and RANKL expression in MG63 cells

OBJECTIVES:To explore the effects of serum from patients with ankylosing spondylitis on the canonical Wnt/β-catenin pathway and to assess whether the serum has an osteogenic effect in MG63 cells.METHODS:MG63 cells were cultured with serum from 45 ankylosing spondylitis patients, 30 healthy controls, or 45 rheumatoid arthritis patients. The relative PPARD, fra-1, MMP7, OPG and RANKL mRNA levels were measured using quantitative real-time polymerase chain reaction. Associations between gene expression and patient demographics and clinical assessments were then analyzed.RESULTS:MG63 cells treated with serum from ankylosing spondylitis patients had higher PPARD, fra-1, MMP7 and OPG gene expression than did cells treated with serum from controls or rheumatoid arthritis patients (all p<0.05). RANKL expression was higher in MG63 cells treated with serum from patients with ankylosing spondylitis or rheumatoid arthritis than in those treated with serum from controls (both p<0.05). The OPG/RANKL ratio was also higher in MG63 cells treated with serum from ankylosing spondylitis patients than in those treated with serum from controls (p<0.05). No associations were found between the expression of the five genes and the patient demographics and clinical assessments (all p>0.05).CONCLUSIONS: Serum from ankylosing spondylitis patients increases PPARD, fra-1, MMP7, OPG and RANKL expression and the OPG/RANKL ratio in MG63 cells; these effects may be due to the stimulatory effect of the serum on the Wnt pathway.

Tooth eruption: altered gene expression in the dental follicle of patients with cleidocranial dysplasia

The dental follicle plays an important role in tooth eruption by providing key regulators of osteogenesis and bone resorption. Patients with cleidocranial dysplasia (CCD) exhibit delayed tooth eruption in combination with increased bone density in the maxilla and mandible, suggesting disturbances in bone remodeling. The aim of this study was to determine the expression of genes relevant for tooth eruption and bone remodeling in the dental follicles of patients with CCD and normal subjects. Thirteen dental follicles were isolated from five unrelated patients with CCD, and fourteen dental follicles were obtained from 10 healthy individuals. All teeth were in the intraosseous phase of eruption. The expression of RANK, RANKL, OPG, and CSF-1 was determined by quantitative RT-PCR. In patients with CCD, the mRNA levels of RANK, OPG, and CSF-1 were significantly elevated compared with the control group. Accordingly, the ratios of RANKL/OPG and RANKL/RANK mRNAs were significantly decreased in patients with CCD. The observed alterations in the expression and ratios of the aforementioned factors in the dental follicle of CCD individuals suggest a disturbed paracrine signaling for bone remodeling that could be responsible for the impaired tooth eruption seen in these patients.