Expression Of Runt-related Transcription Factor Research Articles

Vaspin antagonizes high fat-induced bone loss in rats and promotes osteoblastic differentiation in primary rat osteoblasts through Smad-Runx2 signaling pathway

BackgroundVisceral adipose tissue-derived serine protease inhibitor (vaspin), an adipose-derived hormone, exhibits various biological functions. Recently, studies showed that vaspin is closely related to bone metabolism. However, how vaspin influences bone formation and its underlying mechanisms in high fat-induced obese rats and rat primary osteoblasts (OBs) are not fully understood. In this study, the effects of vaspin on bone mechanical parameters and microarchitecture were evaluated.MethodsA total of 40 male Sprague-Dawley (SD) rats at 5-week old were fed with high fat diet (HFD) and normal diet (ND) for 12 weeks followed by treatment of vaspin for 10 weeks. Micro CT and three-point bending tests were conducted to evaluate bone microstructure and biomechanics. The alkaline phosphatase (ALP) activity, expression of Runt-related transcription factor 2 (Runx2), Osterix (Osx), Collegen alpha1 (Colla1) procollagen I N-terminal peptide (PINP), C-telopeptide of type I collagen (CTX), Smad2/3 and p-Smad2/3 was detected by different methods.ResultsOur data indicated that, compared with ND rats, HFD rats exhibited high body weight, decreased bone strength and deteriorative bone quality. In contrast, vaspin reduced the body weight, improved the whole body metabolic status, enhanced bone strength, trabecular bone mass, and expression of Runx2, Osx, PINP, and decreased the expression level of plasma CTX. In vitro studies showed that vaspin promoted osteogenic differentiation and ALP activity in rat primary OBs in a dose dependent manner. Vaspin also upregulated mRNA expression of osteogenesis-related genes Runx2, Osx and Colla1 and protein expression of Runx2, Smad2/3 and p-Smad2/3.ConclusionsOur results indicated that vaspin protects against HFD-induced bone loss, and promotes osteogenic differentiation by activating the Smad2/3-Runx2 signaling pathway.

Regulatory Effects of O-GlcNAcylation in Vascular Smooth Muscle Cells on Diabetic Vasculopathy

Vascular complications from uncontrolled hyperglycemia are the leading cause of death in patients with diabetes mellitus. Previous reports have shown a strong correlation between hyperglycemia and vascular calcification, which increases mortality and morbidity in individuals with diabetes. However, the precise underlying molecular mechanisms of hyperglycemia-induced vascular calcification remain largely unknown. Transdifferentiation of vascular smooth muscle cells (VSMC) into osteoblast-like cells is a known culprit underlying the development of vascular calcification in the diabetic vasculature. Pathological conditions such as high glucose levels and oxidative stress are linked to enhanced osteogenic differentiation of VSMC both in vivo and in vitro. It has been demonstrated that increased expression of runt-related transcription factor 2 (Runx2), a bone-related transcription factor, in VSMC is necessary and sufficient for the induction of VSMC calcification. Addition of a single O-linked β-N-acetylglucosamine (O-GlcNAc) moiety to the serine/threonine residues of target proteins (O-GlcNAcylation) has been observed in the arteries of diabetic patients, as well as in animal models in association with the enhanced expression of Runx2 and aggravated vascular calcification. O-GlcNAcylation is a dynamic and tightly regulated process, that is mediated by 2 enzymes, O-GlcNAc transferase and O-GlcNAcase. Glucose is metabolized into UDP-β-D-N-acetylglucosamine, an active sugar donor of O-GlcNAcylation via the hexosamine biosynthetic pathway. Overall increases in the O-GlcNAcylation of cellular proteins have been closely associated with cardiovascular complications of diabetes. In this review, the authors provide molecular insights into cardiovascular complications, including diabetic vasculopathy, that feature increased O-GlcNAcylation in people with diabetes.

Interactive effects of dietary vitamin K3 and Bacillus subtilis PB6 on the growth performance and tibia quality of broiler chickens with sex separate rearing

Both vitamin K and probiotics can promote the bone health of poultry and mammals. The present study was conducted to investigate the interactive effects between vitamin K3 (VK3) and Bacillus subtilis PB6 on the growth performance and tibia quality of broiler chickens with sex separate rearing. In a 3 × 2 × 2 factorial arrangement, 720 one-day-old broiler chicks (Arbor Acres) were assigned to 12 groups with three levels of dietary VK3 (0, 0.5 and 4.0 mg/kg), with or without probiotic supplementation (500 g/t) and with sex separation (male and female). Each group included 3 replicates with 20 birds per replicate. During day 1 to 21, 0.5 and 4.0 mg/kg of VK3 increased average daily gain (ADG) of all birds and average daily feed intake of male birds (P < 0.05). During day 22 to 42, probiotic supplementation increased the ADG of birds (P < 0.05). Probiotic addition increased the weight, length, diameter and strength of tibia in all birds, and 0.5 and 4.0 mg/kg of VK3 increased the tibial breaking strength of male birds at day 21 (P < 0.05). Vitamin K3 and probiotic synergistically increased tibial breaking strength at day 42 and ash content at day 21 (P < 0.05). Three factors exhibited interactive effects on the chemical composition of tibia at day 42, and female birds fed 4 mg/kg of VK3 and probiotic had the highest contents of ash, calcium and phosphorus (P < 0.05). Bacillus subtilis PB6 increased the serum phosphorus level of male birds at day 21 and serum calcium level of female ones at day 42 (P < 0.05). At day 21, in the probiotic-supplemented birds, serum osteocalcin (OCN) and bone-specific alkaline phosphatase (BALP) were increased by 0 and 4.0 mg/kg of VK3, respectively (P < 0.05). Probiotic increased serum OCN and cooperated with VK3 to increase the serum BALP at day 42 (P < 0.05). Vitamin K3 and probiotic synergistically down-regulated the mRNA expression of Runt-related transcription factor 2 and OCN at day 21 (P < 0.05). Vitamin K3 down-regulated the alkaline phosphatase (liver/bone/kidney) expression in male birds at day 21 and 42, but probiotic up-regulated the expression of these genes at day 42 (P < 0.05). In conclusion, VK3 and B. subtilis PB6 promoted the growth performance of broilers during starter and grower phases, respectively. They synergistically improved the physical and chemical traits of tibias, especially in grower phase, by modulating calcium and phosphorus metabolism as well as osteogenic gene expression.

Inhibition of CRY2 by STAT3/miRNA-7-5p Promotes Osteoblast Differentiation through Upregulation of CLOCK/BMAL1/P300 Expression

Accumulating evidence indicates that cryptochrome circadian regulatory (CRY) proteins have emerged as crucial regulators of osteogenic differentiation. However, the associated mechanisms are quite elusive. In this study, we show that knockdown of CRY2 downregulated the expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN) to facilitate osteoblast differentiation. Further study identified that CRY2 was directly targeted by microRNA (miR)-7-5p, which was highly induced during osteoblast differentiation. The expression of Runx2, ALP, collagen type I alpha 1 (Col1a1), and OCN was upregulated by overexpression of miR-7-5p and induction of osteoblast differentiation. Moreover, signal transducer and activator of transcription 3 (STAT3) transcriptionally activated miR-7-5p to significantly enhance the expression of above osteogenic marker genes and mineral formation. However, overexpression of CRY2 abolished the osteogenic differentiation induced by miR-7-5p overexpression. Silencing of CRY2 unraveled the binding of CRY2 with the circadian locomotor output cycles kaput (CLOCK)/brain and muscle ARNT-like 1 (BMAL1) complex to release CLOCK/BMAL1, which facilitated the binding of CLOCK/BMAL1 to the promoter region of the P300 E-box to stimulate the transcription of P300. P300 subsequently promoted the acetylation of histone 3 and the formation of a transcriptional complex with Runx2 to enhance osteogenesis. Taken together, our study revealed that CRY2 is repressed by STAT3/miR-7-5p to promote osteogenic differentiation through CLOCK/BMAL1/P300 signaling. The involved molecules may be potentially targeted for treatment of osteoporosis.

Melatonin Attenuates β-Glycerophosphate-Induced Calcification of Vascular Smooth Muscle Cells via a Wnt1/β-Catenin Signaling Pathway.

Background Melatonin has been demonstrated to protect against calcification in cyclosporine nephrotoxicity. The wingless-type MMTV integration site family member 1 (Wnt1)/β-catenin pathway is associated with cardiovascular calcification. This study aimed to explore whether melatonin could attenuate VSMC calcification through regulating the Wnt1/β-catenin signaling pathway. Methods The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualized by Alizarin Red Staining. Calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure the expression of runt-related transcription factor 2 (Runx2), α-smooth muscle actin (α-SMA), and cleaved caspase-3. Results Melatonin markedly ameliorated calcium deposition and ALP activity. Runx2 and cleaved caspase-3 were found to be reduced and α-SMA was found to be increased by melatonin, together with a decrease in apoptosis. Immunofluorescence assay revealed a lower Runx2 protein level in the melatonin group. Melatonin treatment significantly decreased the expression of Wnt1 and β-catenin. Treatment with lithium chloride or transglutaminase 2 abrogated the protective effects of melatonin. Conclusion Melatonin can attenuate β-GP-induced VSMC calcification through the suppression of Wnt1/β-catenin system.

BMP9 promotes osteogenic differentiation of SMSCs by activating the JNK/Smad2/3 signaling pathway.

Synovial mesenchymal stem cells (SMSCs) with high proliferation and multi differentiation ability, and low immunogenicity have attracted research attention for their potential application in tissue engineering. Once their ability of osteogenesis is strengthened, it will be of practical value to apply the SMSCs in the field of bone regeneration. The current study aimed to investigate the osteogenic characteristics of SMSCs induced by bone morphogenetic protein 9 (BMP9) both in vitro and in vivo and to elucidate the mechanism underlying these characteristics. Specifically, different BMPs were assessed to determine the protein that would be the most favorable for stimulating osteogenic differentiation of SMSCs following their separation. The BMP9-enhanced osteogenesis of SMSCs was fully investigated in vitro and in vivo, and the c-Jun N-terminal kinase (JNK)/Smad2/3 signaling pathway stimulated by BMP9 was further explored. Our data suggested that BMP9 could significantly promote gene and protein expression of runt-related transcription factor 2, alkaline phosphatase, osteopontin, and osteocalcin, and SP600125, a JNK-specific inhibitor, could effectively decrease this tendency. Similar results were also confirmed in rats with cranial defects. In conclusion, our study indicated that BMP9 promotes bone formation both in vitro and in vivo possibly by activating the JNK/Smad2/3 signaling pathway.

Effects Of Triptolide On Tooth Movement And Root Resorption In Rats.

PurposeThe aim of this study was to investigate the effects of triptolide on the tooth movement and root resorption in rats during orthodontic treatment.Material and methodsA total of 48 male Wistar rats were divided into three groups of 16 each. The right maxillary first molars of rats were drawn mesially by closed coil nickel–titanium spring with a force of 50 g. The two experimental groups received intraperitoneal injections of triptolide for 14 days at a dose of 15 µg/kg/day and 30 µg/kg/day, respectively. The control group received vehicle injections. After 14 days, the rats were humanely killed. The amount of tooth movement was measured. Eight rats from each group were randomly chosen for analysis of the percentage of root resorption area by scanning electron microscopy. For the remaining eight rats in each group, the H&E staining, tartrate-resistant acid phosphatase (TRAP) staining and immunohistochemistry analysis were performed.ResultsThe amount of tooth movement and the ratio of root resorption area were significantly decreased in the triptolide-treated rats. The number of TRAP-positive cells was significantly lower in triptolide-treated groups. Moreover, the expression of nuclear factor kappa B ligand (RANKL) was reduced. In contrast, the expression of osteoprotegerin was significantly up-regulated. In the tension side, the expressions of runt-related transcription factor 2 and osteocalcin were significantly enhanced by triptolide injection.ConclusionTriptolide injection could arrest orthodontic tooth movement and reduce root resorption in rats via inhibition of osteoclastogenesis. In addition, triptolide may exert a positive effect on osteoblastogenesis.

Uremic serum-induced calcification of human aortic smooth muscle cells is a regulated process involving Klotho and RUNX2.

Vascular calcification (VC) is common in subjects with chronic kidney disease (CKD) and is associated with increased cardiovascular risk. It is an active process involving transdifferentiation of arterial smooth muscle cells (SMCs) into osteogenic phenotype. We investigated the ability of serum from CKD subjects to induce calcification in human SMCs in vitro (calcific potential of sera: CP), and associated changes in expression of Runt-related transcription factor 2 (RUNX2), SM22α, and Klotho. Sera from subjects with CKD (18 stage 3, 17 stage 4/5, and 29 stage 5D) and 20 controls were added to human cultured SMCs and CP quantified. The CP of CKD sera was greater (P<0.01) than that of controls, though not influenced by CKD stage. Modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) (P<0.001), serum phosphate (P=0.042), receptor activator of nuclear factor κappa-B ligand (RANKL) (P=0.001), parathyroid hormone (PTH) (P=0.014), and high-density lipoprotein (HDL)/cholesterol ratio (P=0.026) were independent predictors of CP accounting for 45% of variation. Adding calcification buffer (CB: calcium chloride [7 mM] and β-glycerophosphate [7 mM]) increased the CP of control sera to approximate that of CKD sera. CP of CKD sera was unchanged. CKD sera increased RUNX2 expression (P<0.01) in human SMCs and decreased SM22α expression (P<0.05). Co-incubating control but not CKD serum with CB further increased RUNX2 expression (P<0.01). Both SM22α and Klotho expression decreased significantly (P<0.01) in the presence of CKD serum, and were virtually abolished with stage 5D sera. These findings support active regulation by CKD serum of in vitro VC by induction of RUNX2 and suppression of SM22α and Klotho.

Therapeutic inhibition of microRNA-34a ameliorates aortic valve calcification via modulation of Notch1-Runx2 signalling.

Calcific aortic valve stenosis (CAVS) is the most common valvular heart disease and is increased with elderly population. However, effective drug therapy has not been established yet. This study aimed to investigate the role of microRNAs (miRs) in the development of CAVS. We measured the expression of 10 miRs, which were reportedly involved in calcification by using human aortic valve tissue from patients who underwent aortic valve replacement with CAVS or aortic regurgitation (AR) and porcine aortic valve interstitial cells (AVICs) after treatment with osteogenic induction medium. We investigated whether a specific miR-inhibitor can suppress aortic valve calcification in wire injury CAVS mice model. Expression of miR-23a, miR-34a, miR-34c, miR-133a, miR-146a, and miR-155 was increased, and expression of miR-27a and miR-204 was decreased in valve tissues from CAVS compared with those from AR. Expression of Notch1 was decreased, and expression of Runt-related transcription factor 2 (Runx2) was increased in patients with CAVS compared with those with AR. We selected miR-34a among increased miRs in porcine AVICs after osteogenic treatment, which was consistent with results from patients with CAVS. MiR-34a increased calcium deposition in AVICs compared with miR-control. Notch1 expression was decreased, and Runx2 expression was increased in miR-34a transfected AVICs compared with that in miR-control. Conversely, inhibition of miR-34a significantly attenuated these calcification signals in AVICs compared with miR-control. RNA pull-down assay revealed that miR-34a directly targeted Notch1 expression by binding to Notch1 mRNA 3' untranslated region. In wire injury CAVS mice, locked nucleic acid miR-34a inhibitor suppressed aortic velocity, calcium deposition of aortic valves, and cardiac hypertrophy, which were involved in decreased Runx2 and increased Notch1 expressions. miR-34a plays an important role in the development of CAVS via Notch1-Runx2 signalling pathway. Inhibition of miR-34a may be the therapeutic target for CAVS.

MiR-135-5p promotes osteoblast differentiation by targeting HIF1AN in MC3T3-E1 cells

BackgroundMicroRNAs (miRNAs or miRs) serve crucial roles in the progression of osteoporosis. This study investigated the role and specific molecular mechanism of miR-135-5p in regulating osteoblast differentiation and calcification.MethodsBone morphogenetic protein 2 (BMP2) was employed to interfere with the differentiation of MC3T3-E1. Then, miR-135-5p mimic or miR-135-5p inhibitor was transfected into MC3T3-E1, and quantitative RT-PCR was used to measure the expression of miR-135-5p. The expressions of runt-related transcription factor 2 (Runx2), osterix (OSX), osteopontin (OPN), and osteocalcin (OCN) were determined using western blot. Alkaline phosphatase (ALP) activity was measured using an appropriate kit assay. Calcium nodule staining was evaluated with alizarin red staining. A luciferase reporter assay was used to verify the target of miR-135-5p. Hypoxia-inducible factor 1 α inhibitor (HIF1AN) overexpression was applied to investigate its own role in the mechanism and a miR-135-5p rescue experiment was also performed.ResultsOverexpression of miR-135-5p promoted osteogenic differentiation and calcification, as shown by the increase in ALP activity, calcification and osteogenic marker levels, including Runx2, OSX, OPN and OCN. Knockdown of miR-135-5p yielded the opposite results. HIF1AN was confirmed as a direct target of miR-135-5p. HIF1AN overexpression inhibited osteogenic differentiation and calcification while miR-135-5p reversed these effects.ConclusionsThese results indicate that miR-135-5p might have a therapeutic application related to its promotion of bone formation through the targeting of HIF1AN.

Leonurussibiricus L. ethanol extract promotes osteoblast differentiation and inhibits osteoclast formation.

Leonurus sibiricus L. (LS) is a medicinal plant used in East Asia, Europe and the USA. LS is primarily used in the treatment of gynecological diseases, and recent studies have demonstrated that it exerts anti-inflammatory and antioxidant effects. To the best of our knowledge, the present study demonstrated for the first time that LS may promote osteoblast differentiation and suppress osteoclast differentiation in vitro, and that it inhibited lipopolysaccharide (LPS)-induced bone loss in a mouse model. LS was observed to promote the osteoblast differentiation of MC3T3-E1 cells and upregulate the expression of runt-related transcription factor 2 (RUNX2), a key gene involved in osteoblast differentiation. This resulted in the induction of the expression of various osteogenic genes, including alkaline phosphatase (ALP), osteonectin (OSN), osteopontin (OPN), type I collagen (COL1) and bone sialoprotein (BSP). LS was also observed to inhibit osteoclast differentiation and bone resorption. The expression levels of nuclear factor of activated T-cells 1 (NFATc1) and c-Fos were inhibited following LS treatment. NFATc1 and c-Fos are key markers of osteoclast differentiation that inhibit receptor activator of nuclear factor-κB ligand (RANKL)-induced mitogen-activated protein kinase (MAPKs) and nuclear factor (NF)-κB. As a result, LS suppressed the expression of osteoclast-associated genes, such as matrix metallopeptidase-9 (MMP-9), cathepsin K (Ctsk), tartrate-resistant acid phosphatase (TRAP), osteoclast-associated immunoglobulin-like receptor (OSCAR), c-src, c-myc, osteoclast stimulatory transmembrane protein (OC-STAMP) and ATPase H+ transporting V0 subunit d2 (ATP6v0d2). Consistent with the in vitro results, LS inhibited the reduction in bone mineral density and the bone volume/total volume ratio in a mouse model of LPS-induced osteoporosis. These results suggest that LS may be a valuable agent for the treatment of osteoporosis and additional bone metabolic diseases.

Hypoxia-responsive miRNA-21-5p inhibits Runx2 suppression by targeting SMAD7 in MC3T3-E1 cells.

Sustained hypoxia inhibits osteogenesis and osteoblast differentiation by downregulating the expression of runt‐related transcription factor 2 (Runx2). MicroRNAs (miRNAs) have been shown to regulate osteogenesis and osteoblast differentiation. In the present study, we profiled miRNAs, with microRNA array and quantitative real‐time polymerase chain reaction (RT‐PCR) methods, in mouse osteoblast (MC3T3‐E1) cells under hypoxia. Then, we investigated regulation by miRNA‐21‐5p on the expression of Runx2 and other osteoblast differentiation‐associated markers via gain‐of‐function and loss‐of‐function strategies. We found that expression of miRNA‐21‐5p, miRNA‐210‐5p, and other eight miRNAs was upregulated significantly in hypoxia‐treated MC3T3‐E1 cells. miRNA‐21‐5p overexpression downregulated the expression of the mRNA and protein of suppressor of mothers against decapentaplegic (SMAD7) markedly, the 3′‐untranslated region (3′‐UTR) of which was highly homologous with the miRNA‐21‐5p sequence. miRNA‐21‐5p overexpression upregulated the protein expression of Runx2 in hypoxia‐treated MC3T3‐E1 cells, although mRNA expression of Runx2 and other osteoblast differentiation‐associated molecules (eg, osteocalcin, procollagen type 1 amino‐terminal propeptide, P1NP) were not regulated by it; such upregulation was SMAD7‐dependent. In conclusion, hypoxia‐responsive miRNA‐21‐5p promoted Runx2 expression (at least in part) by targeting the 3′‐UTR and downregulating SMAD7 expression. Our study suggests a protective role of miRNA‐21‐5p in promoting osteoblast differentiation under hypoxia.

TiO2 nanotubes promote osteogenic differentiation of mesenchymal stem cells via regulation of lncRNA CCL3-AS

Titanium (Ti) is widely used as orthopedic implant materials. TiO2 nanotubes (TNTs) further improve the bioactivity of Ti, which enhance the osteogenic differentiation of mesenchymal stem cells (MSCs). However, the underlying mechanism is still unclear. In this study, we verified the response of MSCs on Ti and TNT substrates and explored the regulatory mechanism of long non-coding RNAs (lncRNAs). LncRNA and mRNA expression profiles were analyzed via RNA sequencing. Differential lncRNA and mRNA expression and predicted target genes of lncRNAs were performed by bioinformatics analysis. 1075 up-regulated and 1301 down-regulated genes, 26 up-regulated and 35 down-regulated lncRNAs were obtained according to the RNA-Seq. Expression of 8 lncRNAs were verified by qPCR, which was consistent with the sequencing data. To explore the function and target gene of lncRNA, lncRNA CCL3-AS and gene CCL3 were selected for further investigation. The fluorescence staining, alkaline phosphatase (ALP) activity and CCK-8 assay were performed. Besides, expressions of runt-related transcription factor 2 (Runx2), collagen type I (Col I), osteopontin (OPN) were detected by qPCR and western blot. These results indicate that lncRNA CCL3-AS could inhibit the osteogenic differentiation and enhance cell viability of MSCs on the TNT substrates, which was dependent on the regulation of CCL3. This study supplied a comprehensive understanding for further study using lncRNA modulators to surface design of titanium for enhancing osseointegration.

Complement activation causes oncogene expression in Gaucher disease

Abstract GBA1 mutations lead to defective lysosomal glucocerebrosidase resulting in accumulation of glucosylceramide (GC) in Gaucher disease (GD). Patients with GD have an increased risk to develop B cell lymphomas. The exact mechanistic bases for this propensity remain elusive. Recently, we uncovered formation of GC-specific IgG autoantibodies in Gba1 D409V/knockout (Gba19V/−) mice, which recapitulate features of human GD, and in humans with untreated GD. In vivo formation of IgG-GC immune complexes induced massive complement activation and C5a generation. Importantly, C5a-mediated activation of its cognate C5a receptor 1 (C5aR1) on immune cells enhanced GC synthesis, thereby fueling GC accumulation and excess tissue recruitment and activation of inflammatory myeloid and lymphoid immune cells, leading to visceral tissue damage in GD. Here, the expression of Runt-related transcription factor 1 (RUNX-1) was determined in Gba19V/− mice, to evaluate if C5a/C5aR1 axis activation may control the development of lymphomas in GD. RUNX-1 is a member of the Runt oncogene family linked to hematologic malignancies. We determined RUNX-1 expression in tissue from C5aR1 sufficient (+/+) and deficient (−/−) Gba19V/− mice as well as strain-matched control WT and C5aR1−/− mice. Compared to WT, Gba19V/− mice had increased RUNX-1 expression. Strikingly, RUNX-1 expression was markedly downregulated in C5aR−/−Gba19V/− vs. C5aR1+/+Gba19V/− mice. Our findings suggest that the C5a-C5aR1 axis activation in GD drives RUNX1 expression as a novel mechanism to control the development of hematologic malignancies in GD that may be diminished by targeting the C5aR1 axis in GD.

Identification and characterization of a polysaccharide from the roots of Morinda officinalis, as an inducer of bone formation by up-regulation of target gene expression

Morinda officinalis is an important traditional tonic herbal medicine. In the present study, we found that crude polysaccharides extracted from M. officinalis, named MO90, could significantly increase the bone mineral density (BMD) of the whole femur, distal femur, and proximal femur in ovariectomized (OVX) rats. In addition, MO90 decreased the level of bone turnover markers and prevented the deterioration of trabecular microarchitecture. To investigate the fractions responsible for anti-osteoporosis activity, one novel inulin-type fructan, MOW90-1, was isolated from MOP90. Structural analysis indicated that MOW90-1 consists of a backbone of (2→1)-linked-β-D-Fruf, and is terminated with (1→)-linked-α-D-Glcp and (2→)-linked-β-D-Fruf. Furthermore, an in vitro anti-osteoporosis assay indicated that MOW90-1 promoted proliferation, differentiation, and mineralization of MC3T3-E1 cells by up-regulating the expression of runt-related transcription factor 2, osterix, osteopontin, and osteocalcin. In conclusion, our studies provide supporting evidence for future use of this novel M. officinalis fructan as a key nutrient of health products.

Effect of sclerostin on the functions and related mechanisms of cementoblasts under mechanical stress

The purpose of this study is to investigate the potential effects of sclerostin (SOST) on the biological funtions and related mechanisms of cementoblasts under mechanical stress. OCCM-30 cells were treated with varying doses of SOST (0, 25, 50, and 100 ng·mL⁻¹) and were loaded with uniaxial compressive stress (2 000 μ strain with a frequency of 0.5 Hz) for six hours. Western blot was utilized to detect the expressions of β-catenin, p-smad1/5/8, and smad1/5/8 proteins. Alkaline phosphatase (ALP) activity was determined, and reverse transcription polymerase chain reaction was used to measure the expressions of runt-related transcription factor 2 (Runx-2), osteocalcin (OCN), bone sialoproteins (BSP), receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) mRNA. The expression of p-smad 1/5/8 was significantly downregulated with increasing SOST. β-catenin and smad1/5/8 exhibited no difference. ALP activity decreased under mechanical compressive stress with increasing SOST concentrations. Runx-2 expression was reduced with increasing SOST concentrations, and a similar trend was observed for the BSP and OCN expressions. When the SOST concentration was enhanced, RANKL expression gradually increased, whereas the expression of OPG decreased. Under mechanical comprehensive stress, SOST can adjust the bone morphogenetic protein (BMP) /smad signal pathway. Osteosclerosis inhibits the mineralization of cementoblasts under mechanical compressive stress, which may be achieved by inhibiting the expressions of osteogenesis factors (Runx2, OCN, BSP, and others) and by promoting the ratio of cementoclast-related factors (RANKL/OPG) through BMP signal pathways.

Expressions of runt-related transcription factor 2 and osteopontin in breast cancer tissues and their clinical significance

Expressions of runt-related transcription factor 2 and osteopontin in breast cancer tissues and their clinical significance

High-fat and high-glucose microenvironment decreases Runx2 and TAZ expression and inhibits bone regeneration in the mouse

BackgroundType 2 diabetes mellitus (T2DM) and hyperlipidemia are negatively related to bone regeneration. The aim of this study was to evaluate the effect of high-fat and high-glucose microenvironment on bone regeneration and to detect the expression of runt-related transcription factor 2 (Runx2) and transcriptional co-activator with PDZ-binding domain (TAZ) during this process.MethodsAfter establishing a high-fat and high-glucose mouse model, a 1 mm × 1.5 mm bone defect was developed in the mandible. On days 7, 14, and 28 after operation, bone regeneration was evaluated by hematoxylin-eosin staining, Masson staining, TRAP staining, and immunohistochemistry, while Runx2 and TAZ expression were detected by immunohistochemistry, RT-PCR, and Western blot analysis.ResultsOur results showed that the inhibition of bone regeneration in high-fat and high-glucose group was the highest among the four groups. In addition, the expression of Runx2 in high-fat, high-glucose, and high-fat and high-glucose groups was weaker than that in the control group, but the expression of TAZ only showed a decreasing trend in the high-fat and high-glucose group during bone regeneration.ConclusionsIn conclusion, these results suggest that high-fat and high-glucose microenvironment inhibits bone regeneration, which may be related to the inhibition of Runx2 and TAZ expression.

Amelioration of Uremic Toxin Indoxyl Sulfate-Induced Osteoblastic Calcification by SET Domain Containing Lysine Methyltransferase 7/9 Protein

Background: Vascular calcification (VC) is a very common phenomenon in patients with chronic kidney disease (CKD). It has been reported that some histone methylation play a role in VC as an epigenetic regulator. Indoxyl sulfate (IS) is a protein-bound uremic toxin that has been proven as one of the major risk factors of cardiovascular disease in CKD. SET domain containing lysine methyltransferase 7/9 (SET7/9) is one of the important histone methyltransferases. Objectives: This study aimed to determine the effect of IS on the expression of SET7/9 and the role of SET7/9 in IS-induced osteoblastic differentiation and calcification of vascular smooth muscle cells (VSMCs). Methods: VSMCs were incubated with various concentrations of IS for different durations to assess osteoblastic differentiation and expression of SET7/9. Western blot analysis and quantitative real-time polymerase chain reaction were performed to assess the protein and mRNA levels of SET7/9 respectively. The calcium content was measured to evaluate calcification. Results: Osteoblastic differentiation and calcification of VSMCs and downregulation of the expression of SET7/9 were observed after IS treatment. The autophagy was activated after treatment with IS, whereas the inhibition of the autophagy partially attenuated the effect of IS on both the stimulation of the expression of runt-related transcription factor 2 and calcium deposition. Conclusions: Our data demonstrated that SET7/9 downregulation and autophagy activation may be the key mechanism of IS-induced VC in CKD.

N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide promotes bone formation via the canonical Wnt/β-catenin signaling pathway.

Osteoporosis is characterized by low bone mineral density and microarchitectural deterioration of bone tissue. N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (MBOC) is one of the macamides isolated from Maca (Lepidium meyenii Walp.), a cruciferous plant from the Andes of Peru. In this study, C3H/10T1/2 mesenchymal stem cells were treated with MBOC in osteogenic induction medium. An ovariectomized (OVX) mouse model was used to investigate the effect of 1-month MBOC treatment on the prevention of postmenopausal osteoporosis. Remarkably, trabecular thickness, trabecular number, and bone volume/tissue volume of the distal femoral metaphysis were significantly increased in OVX+MBOC mice compared with OVX mice, as revealed by microcomputed tomography analysis. Trabecular separation was decreased in OVX+MBOC mice compared with OVX mice. Consistently, MBOC increased the levels of osteocalcin and runt-related transcription factor 2 in OVX mice, as well as the expression of runt-related transcription factor 2, osterix, and alkaline phosphatase in C3H/10T1/2 cells. Mechanistically, MBOC activates the canonical Wnt/β-catenin signaling pathway via inhibiting phosphorylation of GSK-3β at Tyr216 and maintaining β-catenin expression. Collectively, the current study demonstrates the robustness of MBOC in the induction of mesenchymal stem cells osteogenic differentiation and consequent bone formation, suggesting that MBOC may be a potentially effective drug to treat postmenopausal osteoporosis.