Inhibits Osteoblast Differentiation Research Articles

Examining the Role of Hypothalamus-Derived Neuromedin-U (NMU) in Bone Remodeling of Rats.

Global loss of the neuropeptide Neuromedin-U (NMU) is associated with increased bone formation and high bone mass in male and female mice by twelve weeks of age, suggesting that NMU suppresses osteoblast differentiation and/or activity in vivo. NMU is highly expressed in numerous anatomical locations including the skeleton and the hypothalamus. This raises the possibility that NMU exerts indirect effects on bone remodeling from an extra-skeletal location such as the brain. Thus, in the present study we used microinjection to deliver viruses carrying short-hairpin RNA designed to knockdown Nmu expression in the hypothalamus of 8-week-old male rats and evaluated the effects on bone mass in the peripheral skeleton. Quantitative RT-PCR confirmed approximately 92% knockdown of Nmu in the hypothalamus. However, after six weeks, micro computed tomography on tibiae from Nmu-knockdown rats demonstrated no significant change in trabecular or cortical bone mass as compared to controls. These findings are corroborated by histomorphometric analyses which indicate no differences in osteoblast or osteoclast parameters between controls and Nmu-knockdown samples. Collectively, these data suggest that hypothalamus-derived NMU does not regulate bone remodeling in the postnatal skeleton. Future studies are necessary to delineate the direct versus indirect effects of NMU on bone remodeling.

Sclerostin blockade promotes bone metastases of Wnt-responsive breast cancer cells.

The secreted protein sclerostin is primarily produced by osteocytes and suppresses osteoblast differentiation and function by inhibiting the canonical Wnt signaling pathway. Genetic and pharmacological inhibition of sclerostin has been shown to increase bone formation and an anti-sclerostin antibody has been clinically approved for the treatment of osteoporosis. Canonical Wnt signaling is also involved in the progression of several types of cancers including breast cancer. Here, we studied the effects of sclerostin inhibition on the development of bone metastases of breast cancer using mouse models. TOPFLASH assay and real-time PCR analysis of AXIN2, a target of canonical Wnt signaling, revealed that, among four cell lines tested, MDA-MB-231 human breast cancer cells responded highly to the canonical Wnt ligand Wnt3a, whereas other cell lines exhibited marginal responses. Consistent with these results, treatment with an anti-sclerostin antibody significantly increased the bone metastases of MDA-MB-231 but not those of other breast cancer cells. Immunohistochemical studies demonstrated that an anti-sclerostin antibody induced intracellular accumulation of β-catenin in bone-colonized MDA-MB-231 cells. Suspension culture assays showed that Wnt3a accelerated the tumorsphere formation of MDA-MB-231 cells, whereas monolayer cell proliferation and migration were not affected. Furthermore, the numbers of osteoclasts and their precursor cells in bone metastases of MDA-MB-231 were significantly increased in mice treated with an anti-sclerostin antibody. These results collectively suggest that sclerostin blockade activates canonical Wnt signaling in ligand-responsive breast cancer cells metastasized to bone, thereby increasing bone metastases, likely to have been mediated at least in part by enhancing stem cell-like properties of cancer cells and osteoclastogenesis.

Ellagic acid inhibits CDK12 to increase osteoblast differentiation and alleviate osteoporosis in hindlimb-unloaded and ovariectomized mice

BackgroundOsteoporosis is a highly prevalent bone disease occurred commonly in astronauts and postmenopausal women due to mechanical unloading and estrogen deficiency, respectively. At present, there are some traditional Chinese medicine compounds for preventing and treating osteoporosis induced by simulated microgravity, but the detailed components of the traditional Chinese medicines still need to be confirmed and osteoporosis is still untreatable due to a lack of effective small-molecule natural medicine. PurposeTo explore the role of cyclin-dependent kinase 12 (CDK12) in osteoporosis induced by simulated microgravity and the therapeutic effect of CDK12-targeted Ellagic Acid (EA) on osteoporosis. MethodsOur previous study has suggested that CDK12 as a potential target for treating and preventing osteoporosis. In this study, the role of CDK12 in osteoblasts and mice bone tissues was further studied under simulated microgravity. And by targeting CDK12, natural small-molecule product EA was screened out based on a large scale through the weighted set similarity (WES) method and the therapeutic effects of EA on osteoporosis was investigated in hindlimb-unloaded (HU) mouse model and ovariectomized (OVX) model. ResultsThe results demonstrated that simulated microgravity inhibited bone formation and up-regulated the expression of CDK12. Furthermore, CDK12-siRNA or THZ531 (an inhibitor of CDK 12) promoted osteoblast differentiation, while the overexpression of CDK12 inhibited osteoblasts differentiation. And we further proved that CDK12-targeted EA showed a rescue effect on osteoblast differentiation inhibition caused by simulated microgravity. EA (50 mg·kg−1·day−1) daily intragastric administration alleviated the symptoms of osteoporosis and accompanied with the improvement of trabecular bone and cortical bone parameters with significantly overexpression of CDK12. ConclusionEA efficiently improves osteoporosis by targeting CDK12, which is a suppresser of osteoblast differentiation and a novel therapeutic target for treating osteoporosis.

Lack of association of superoxide dismutase I/D variant with osteopenia/osteoporosis in Turkish postmenopausal women

Background Osteoporosis (OP) is a common systemic, metabolic bone disease that affects 40% of postmenopausal women. Oxidative stress (OS) ìs caused by reactive oxygen species (ROS), inhibits osteoblast differentiation and causes apoptosis in osteoblastic cells. Superoxide dismutase (SOD) reduces OS by playing a role in the reduction and defense of intracellular ROS. Therefore, the purpose of this study was to investigate the relationship between osteopenia/OP and the SOD1 50-bp insertion/deletion (I/D) variant in Turkish postmenopausal women. Methods A total of 180 women participated in this study includḭng 89 osteopenia/OP postmenopausal women and 91 healthy postmenopausal women. T-score > −1 standard deviation (SD) defined normal bone mass, T-score between −1 and −2.5 SD defined osteopenia, T-score ≥ −2.5 SD was defined as OP. DNA was extracted from all subjects and the SOD1 I/D variant genotyped by PCR. The results of the analyses were evaluated for statistical significance. Results The mean age of 89 osteopenia/OP patients aged 45 to 74 was 58.57 ± 6.57. There was no D/D homozygous genotype in the patient and control groups. The prevalence of genotypes of I/I, and I/D, profiles for the SOD1 I/D variant were 76.4%, and 23.6% respectively, in patients, and 72.5%, and 27.5% respectively, in the control group. When the patient group and control group were compared, the SOD1 I/D genotype distribution and allele frequencies did not show a significant difference between the groups (p > 0.05). Conclusion Our results showed that the SOD1 I/D variant may not be considered a determining factor in the development of osteopenia/OP in a Turkish population sample. However, ethnic differences, gene-gene, and gene-environment interactions should not be ignored.

Overexpression of Wnt7b antagonizes the inhibitory effect of dexamethasone on osteoblastogenesis of ST2 cells.

It is well established that glucocorticoid-induced osteoporosis is highly associated with preosteoblast differentiation and function. This study is based on the premise that Wnt7b can promote bone formation through Wnt signalling pathway because it can stimulate preosteoblast differentiation and increase its activity. However, it is unknown whether Wnt7b can rescue the inhibited osteoblast differentiation and function caused by exogenous glucocorticoid. In this study we used Wnt7b overexpression ST2 cells to explore whether Wnt7bcan rescue the inhibited osteoblast differentiation and function, which can provide strong proof to investigate a new drug for curing the glucocorticoid induced osteoporosis. We found that Wnt7b can rescue the suppressed osteoblast differentiation and function without cell viability caused by dexamethasone.

Exosomes from LSD1 knockdown breast cancer cells activate osteoclastogenesis and inhibit osteoblastogenesis

Bone metastasis is a common and incurable complication of breast cancer. Lysine-specific demethylase 1 (LSD1), a histone demethylase, plays an important role in the metastasis of breast cancer. However, the role of LSD1 in bone metastasis of breast cancer is unclear. We hypothesized that exosomes from LSD1 knockdown breast cancer cells promote bone metastasis by remodeling bone microenvironment. To verify this hypothesis, exosomes from LSD1 knockdown Estrogen receptor-positive cancer cell lines, MCF7 and T47D, were isolated, and the effects of these exosomes on osteoblast and osteoclast differentiation were investigated. Interestingly, exosomes from LSD1 knockdown breast cancer cells inhibited osteoblast differentiation and promoted osteoclast differentiation. Mechanistically, miR-6881-3p was decreased in the exosomes from LSD1 knockdown cells, and miR-6881-3p suppressed the expression of pre-B-cell leukemia homeobox 1 (PBX1) and additional sex combs like-2 (ASXL2), two genes with essential functions in osteoblast and osteoclast differentiations respectively. Transfection of miR-6881-3p into LSD1 knockdown cells reversed the effects of the exosomes on osteoblast and osteoclast differentiations. Our study reveals important roles of LSD1 on the regulation of exosomal miRNAs and the formation of favorable bone microenvironment for metastasis.

Erythromycin Restores Osteoblast Differentiation and Osteogenesis Suppressed by Porphyromonas gingivalis Lipopolysaccharide.

The macrolide erythromycin (ERM) inhibits excessive neutrophil accumulation and bone resorption in inflammatory tissues. We previously reported that the expression of developmental endothelial locus-1 (DEL-1), an endogenous anti-inflammatory factor induced by ERM, is involved in ERM action. Furthermore, DEL-1 is involved in the induction of bone regeneration. Therefore, in this study, we investigated whether ERM exerts an osteoblastogenic effect by upregulating DEL-1 under inflammatory conditions. We performed in vitro cell-based mechanistic analyses and used a model of Porphyromonas gingivalis lipopolysaccharide (LPS)-induced periodontitis to evaluate how ERM restores osteoblast activity. In vitro, P. gingivalis LPS stimulation suppressed osteoblast differentiation and bone formation. However, ERM treatment combined with P. gingivalis LPS stimulation upregulated osteoblast differentiation-related factors and Del1, indicating that osteoblast differentiation was restored. Alveolar bone resorption and gene expression were evaluated in a periodontitis model, and the results confirmed that ERM treatment increased DEL-1 expression and suppressed bone loss by increasing the expression of osteoblast-associated factors. In conclusion, ERM restores bone metabolism homeostasis in inflammatory environments possibly via the induction of DEL-1.

Chordin Like-1 Regulates Osteoblast and Adipocyte Differentiation Through Stabilizing Insulin-Like Growth Factor Binding Protein 3.

Chordin like-1 (CHRDL1) is an antagonist of bone morphogenetic proteins (BMPs) that acts through binding BMPs and blocking their interaction with BMP receptors. CHRDL1 plays a role in osteoblast differentiation but controversial effects were reported. On the other hand, the role of CHRDL1 in adipogenesis is unknown. In the present study, we investigated the function of CHRDL1 in regulating differentiation of osteoblasts and adipocytes and elucidated the underlying mechanism. CHRDL1 expression was downregulated during osteogenesis while it was upregulated during adipogenesis in primary cultured and established mesenchymal progenitor cell lines. Functional experiments revealed that CHRDL1 suppressed osteoblast differentiation and promoted adipocyte differentiation. Mechanistic explorations revealed that CHRDL1 is directly bound to insulin-like growth factor binding protein 3 (IGFBP3) and attenuated the degradation of the latter. Furthermore, CHRDL1 and IGFBP3 suppressed the activity of insulin receptor substrate 1 (IRS1)/AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin kinase complex 1 (mTORC1) signaling in progenitor cells undergoing osteogenic differentiation. By contrast, they activated AKT/mTORC1 signaling independently of IRS1 during adipogenic differentiation. CHRDL1 enhanced the interaction of nuclear IGFBP3 and retinoid X receptor α (RXRα) during adipogenesis, and inhibition of RXR inactivated AKT and attenuated the stimulation of adipogenic differentiation by CHRDL1. Overexpression of IGFBP3 relieved the perturbation of osteogenic and adipogenic differentiation of progenitor cells induced by CHRDL1 silencing. Finally, CHRDL1 and IGFBP3 were upregulated in the trabecular bone of aged mice. Our study provides evidence that CHRDL1 reciprocally regulates osteoblast and adipocyte differentiation through stabilizing IGFBP3 and differentially modulating AKT/mTORC1 signaling.

CircStrn3 targeting microRNA-9-5p is involved in the regulation of cartilage degeneration and subchondral bone remodelling in osteoarthritis

Circular RNA (circRNA) is involved in the regulation of articular cartilage degeneration induced by inflammatory factors or oxidative stress. In a previous study, we found that the expression of circStrn3 was significantly reduced in chondrocytes of osteoarthritis (OA) patients and OA mice. Therefore, the aim of this paper was to explore the role and mechanism of circStrn3 in osteoarthritis. Minus RNA sequencing, fluorescence in situ hybridization, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of circStrn3 in human and mouse OA cartilage tissues and chondrocytes. Chondrocytes were then stimulated to secrete exosomal miR-9-5p by cyclic tensile strain. Intra-articular injection of exosomal miR-9-5p into the model induced by destabilized medial meniscus (DMM) surgery was conducted to alleviate OA progression. Tensile strain could decrease the expression of circStrn3 in chondrocytes. CircStrn3 expression was significantly decreased in human and mouse OA cartilage tissues and chondrocytes. CircStrn3 could inhibit matrix metabolism of chondrocytes through competitively 'sponging' miRNA-9-5p targeting Kruppel-like factor 5 (KLF5), indicating that the decrease in circStrn3 might be a protective factor in mechanical instability-induced OA. The tensile strain stimulated chondrocytes to secrete exosomal miR-9-5p. Exosomes with high miR-9-5p expression from chondrocytes could inhibit osteoblast differentiation by targeting KLF5. Intra-articular injection of exosomal miR-9-5p alleviated the progression of OA induced by destabilized medial meniscus surgery in mice. Taken together, these results demonstrate that reduction of circStrn3 causes an increase in miR-9-5p, which acts as a protective factor in mechanical instability-induced OA, and provides a novel mechanism of communication among joint components and a potential application for the treatment of OA.Cite this article: Bone Joint Res2023;12(1):33-45.

MLK3 silence suppressed osteogenic differentiation and delayed bone formation via influencing the bone metabolism and disturbing MAPK signaling.

Mixed lineage kinase 3 (MLK3) is a member of a serine/threonine MAP3K family, and it has been demonstrated to play critical roles in various biological activities and disease progression. Previous studies showed that impaired skeletal mineralization and spontaneous tooth fracture in the MLK3-deficient mice, suggesting MLK3 actively participated in the bone formation. However, the detailed function and underlying mechanisms remain obscure. The MLK3 knockout (KO) mouse was applied in the present study, and multi-omics were performed to compare the metabolites and gene expression between wild type (WT) and KO mice. The bone fracture model was successfully established, and the healing process was evaluated by X-ray, micro-CT examination, histomorphometry and immunohistochemistry (IHC) staining. On the other hand, the effects of MLK3 on osteogenic differentiation were assessed by alkaline phosphatase (ALP) activity, Alizarin red S (ARS) staining and qRT-PCR examination. Finally, the downstream signaling pathways were screened out by RNA-sequencing (RNA-seq) and then validated by Western blotting. In the present study, imbalanced bone metabolism was observed in these MLK3 KO mice, suggesting MLK3 may participate in bone development. Moreover, MLK3 -/- mice displayed abnormal bone tissues, impaired bone quality, and delayed fracture healing. Further investigation showed that the inhibition of MLK3 attenuated osteoblast differentiation in vitro. According to the RNA-seq data, MAPK signaling was screened out to be a downstream pathway, and its subfamily members extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal protein kinase (JNK) were subjected to Western blotting examination. The results revealed that although no differences in their expression were observed between MSCs derived from WT and KO mice, their phosphorylated protein levels were all suppressed in MLK3 -/- MSCs. In conclusion, our results demonstrated that loss of MLK3 suppressed osteoblast differentiation and delayed bone formation via influencing metabolism and disturbing MAPK signaling. The findings based on the current study demonstrated that MLK3 promoted osteogenesis, stimulated new bone formation and facilitated fracture healing, suggesting that MLK3 may serve as a potential therapeutic target for bone regeneration. MLK3 activator therefore may be developed as a therapeutic strategy for bone fracture.

The effects of carisoprodol on endochondral ossification: A review of the literature and implications for bone health

Carisoprodol is a medication commonly prescribed for musculoskeletal pain, but recent studies have raised concerns about its potential negative effects on bone development and health, particularly in relation to endochondral ossification. Endochondral ossification is a critical process that involves the transformation of cartilage into bone, which is essential for the formation of long bones in the body. Carisoprodol has been shown to reduce the activity of osteoblasts while increasing the activity of osteoclasts, leading to an imbalance in bone formation and resorption. Studies also suggest that carisoprodol may inhibit osteoblast differentiation, decrease bone density, strength, and microarchitecture, and affect the expression of genes involved in endochondral ossification. These negative effects may be due, in part, to its inhibition of the Wnt signaling pathway. Healthcare providers should carefully consider the potential risks of carisoprodol on bone development and health when prescribing this medication. Alternative treatments may be considered for patients at high risk of bone-related complications.

SOX2 suppresses osteoblast differentiation of MC3T3-E1 cells through activating the transcription of LGR4.

Osteogenic differentiation is a crucial process of new bone formation. This study aimed to explore the roles and mechanism of SRY-Box Transcription Factor 2 (SOX2) on proliferation and osteogenic differentiation of MC3T3-E1 cells. Bone morphogenetic protein 2 (BMP2) was used to induce the osteogenic differentiation of MC3T3-E1 cells. The expression of SOX2 was determined by quantitative real-time PCR (RT-PCR) at different time points after induction. The SOX2 overexpression plasmids were constructed and transfected into MC3T3-E1 cells. Osteogenic differentiation was evaluated by Alizarin Red S staining and alkaline phosphatase (ALP) assay. The expressions of osteogenic differentiation markers including runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) were detected by western blot assay. Luciferase reporter and CHIP assays were used to confirm that SOX2 regulated the transcriptional activation of leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4). We found that SOX2 was down-regulated upon BMP2-induced osteogenic differentiation in MC3T3-E1 cells. Overexpression of SOX2 effectively inhibited osteogenic differentiation with decreased ALP activity, calcification, and osteogenic differentiation markers' expression including Runx2, OPN, and OCN. LGR4 was identified as a target of SOX2, and the inhibitory effect of SOX2 on osteogenic differentiation was reversed by knockdown of LGR4. The present study confirmed that SOX2 suppressed osteogenic differentiation of MC3T3-E1 cells through targeting LGR4, which possesses a therapeutic strategy for bone formation and generation.

Metabolic switching and cell wall remodelling of Mycobacterium tuberculosis during bone tuberculosis

ObjectivesBone tuberculosis (TB) is the third most common types of extrapulmonary tuberculosis. It is critical to understand mycobacterial adaptive strategies within bone lesions to identify mycobacterial factors that may have role in disease pathogenesis. MethodsWhole genome microarray was used to characterize the in-vivo transcriptome of Mycobacterium tuberculosis (M.tb) within bone TB specimens. Mycobacterial virulent proteins were identified by bioinformatic software. An in vitro osteoblast cell line model was used to study the role of these proteins in bone TB pathogenesis. Results914 mycobacterial genes were significantly overexpressed and 1688 were repressed in bone TB specimens. Pathway analysis of differentially expressed genes demonstrated a non-replicative and hypometabolic state of M.tb, reinforcement of the mycobacterial cell wall and induction of DNA damage repair responses, suggesting possible survival strategies of M.tb within bone. Bioinformatics mining of microarray data led to identification of five virulence proteins. The genes encoding these proteins were also upregulated in the in vitro MC3T3 osteoblast cell line model of bone TB. Further, exposure of osteoblast cells to two of these virulence proteins (Rv1046c and Rv3663c) significantly inhibited osteoblast differentiation. ConclusionM.tb alters its transcriptome to establish infection in bone by upregulating certain virulence genes which play a key role in disturbing bone homeostasis.

Study on the Mechanism of miR-146a in Gingival Mesenchymal Stem Cells.

This study aimed to investigate the molecular mechanisms of microRNA-146a (miR-146a) on gingival mesenchymal stem cells (MSCs). Gingival MSCs were isolated from the gingiva tissues of patients with periodontal disease to reveal the function of miR-146a in regulating osteoblast differentiation. miR-146a inhibits osteoblast differentiation by inhibiting phosphorylated cyclic-AMP response binding (CREB) protein translocation into the nucleus and ultimately attenuating runt-related transcription factor 2 (Runx2) expression. Furthermore, silencing miR-146a promotes the proliferation of gingival MSCs. Of note, targeted inhibition of miR-146a also inhibited LPS-induced inflammatory response and promoted the proliferation of gingival MSCs via CREB/Runx2 axis. MiR-146a is a key negative regulator of gingival MSCs proliferation and osteogenic differentiation, and targeting to reduce the miR-146a expression is essential for bone formation signaling. Therefore, we propose that miR-146a is a useful therapeutic target for the development of bone anabolic strategies.

DIFFERENCES IN OSTEOBLAST COUNT AND ITS EFFECT ON ORTHODONTIC MOVEMENT OF DIABETIC MODELS’ TEETH AFTER ORAL ADMINISTRATION OF OLIVE OIL

Background: In patients with uncontrolled diabetes, an increase in Advanced glycation end products (AGEs) inhibits osteoblast differentiation so that the number of osteoblasts decreases and interferes with bone formation in orthodontic movement. Giving natural ingredients such as olive oil is proven to increase the number of osteoblasts. Purpose: To determine differences in the number of osteoblasts and their effect on the orthodontic movement of diabetic teeth after oral administration of olive oil. Method: 25 male guinea pigs divided into four groups: diabetic guinea pigs treated with olive oil at a dose of 0.7; 1.05; 1.4 ml/day, distilled water, and healthy guinea pigs were given aquadest. Alveolar bone tissue samples were taken on the 15th day after orthodontic application, and then the number of osteoblasts was calculated histologically. Result: In the diabetic model group treated with olive oil doses of 0.7; 1.05; 1.4 ml/day, distilled water, and healthy guinea pigs given aquades were 2.900 ± 0.678; 4.320 ± 0.497; 5.920 ± 0. 460; 0.200 ± 0.200; 1.320 ± 0.415. The study showed that the olive oil treatment group had more osteoblasts than the untreated group. Analysis one way ANOVA showed a very significant difference between the treatment group and the untreated group (p≤0.01), while the LSD test showed a significant difference between groups. The highest number of osteoblasts was found at a dose of 1.4 ml/day. Conclusion: There was an increase in osteoblasts in the orthodontic movement of diabetic teeth after oral administration of olive oil.

SIRT6 Prevents Glucocorticoid-Induced Osteonecrosis of the Femoral Head in Rats.

Objective Glucocorticoid-induced osteonecrosis of the femoral head is one of the most common causes of nontraumatic osteonecrosis of the femoral head, but its exact pathogenesis remains unclear. The aim of this study was to investigate the role of SIRT6 in the maintenance of bone tissue morphology and structure, intravascular lipid metabolism, and its potential molecular mechanism in glucocorticoid-induced osteonecrosis of the femoral head. Methods SIRT6 adenovirus was transfected into GIONFH in rats. The microstructure of rat bone was observed by micro-CT and histological staining, and the expression of bone formation-related proteins and angiogenesis-related factors was determined through western blot and immunohistochemistry. Alkaline phosphatase activity, alizarin red staining, and the expression levels of Runx2 and osteocalcin were used to evaluate the osteogenic potential. And in vitro tube formation assay and immunofluorescence were used to detect the ability of endothelial cell angiogenesis. Results Dexamethasone significantly inhibited osteoblast differentiation, affected bone formation, and destroyed microvessel formation, increased the intracellular Fe2+ and ROS levels and induced the occurrence of ferroptosis. SIRT6 can inhibit ferroptosis and restore the ability of bone formation and angiogenesis. Conclusion SIRT6 can inhibit the occurrence of ferroptosis, reduce the damage of vascular endothelium, and promote osteogenic differentiation, so as to prevent the occurrence of osteonecrosis of the femoral head.

Zoledronate promotes osteoblast differentiation in high-glucose conditions via the p38MAPK pathway.

Zoledronate (ZOL) were found to inhibit bone resorption in an animal model of diabetes, high glucose concentrations have been shown to decreased the osteogenesis-related gene expression. But the molecular mechanism by which high glucose levels affect osteoblasts and the effects of ZOL on osteoblast differentiation in a high-glucose environment remain unclear. Therefore, we aimed to investigate the effect of ZOL on osteoblast differentiation in a high-glucose environment and determine the responsible mechanism. Cell proliferation was detected by MTT assay, and cell differentiation was evaluated by immunofluorescence staining for alkaline phosphatase expression, alizarin red staining, cytoskeletal arrangement, and actin fiber formation. Real-time PCR and western blot analyses were performed to detect the mRNA and protein expression of p38MAPK, phosphorylated (p)-p38MAPK, CREB, p-CREB, collagen (COL) I, osteoprotegerin (OPG), and RANKL. The results showed that cell proliferation activity did not differ among the groups. But high glucose inhibited osteoblast differentiation; actin fiber formation; and p38MAPK, p-p38MAPK, CREB, p-CREB, COL I, and OPG expression, while promoting RANKL expression. However, we found that treatment with ZOL reversed these effects of high glucose. And further addition of a p38MAPK inhibitor led to inhibition of osteoblast differentiation and actin fiber formation, and lower p38MAPK, p-p38MAPK, CREB, p-CREB, COL I, and OPG expression than in the high glucose +ZOL group with higher RANKL expression than in the high glucose +ZOL group. Collectively, this study demonstrates that high glucose inhibits the differentiation of osteoblasts, and ZOL could partly overcome these effects by regulating p38MAPK pathway activity.

Cyclophilin A Promotes Osteoblast Differentiation by Regulating Runx2.

Cyclophilin A (CypA) is a ubiquitously expressed and highly conserved protein with peptidyl-prolyl cis-trans isomerase activity that is involved in various biological activities by regulating protein folding and trafficking. Although CypA has been reported to positively regulate osteoblast differentiation, the mechanistic details remain largely unknown. In this study, we aimed to elucidate the mechanism of CypA-mediated regulation of osteoblast differentiation. Overexpression of CypA promoted osteoblast differentiation in bone morphogenic protein 4 (BMP4)-treated C2C12 cells, while knockdown of CypA inhibited osteoblast differentiation in BMP4-treated C2C12. CypA and Runx2 were shown to interact based on immunoprecipitation experiments and CypA increased Runx2 transcriptional activity in a dose-dependent manner. Our results indicate that this may be because CypA can increase the DNA binding affinity of Runx2 to Runx2 binding sites such as osteoblast-specific cis-acting element 2. Furthermore, to identify factors upstream of CypA in the regulation of osteoblast differentiation, various kinase inhibitors known to affect osteoblast differentiation were applied during osteogenesis. Akt inhibition resulted in the most significant suppression of osteogenesis in BMP4-induced C2C12 cells overexpressing CypA. Taken together, our results show that CypA positively regulates osteoblast differentiation by increasing the DNA binding affinity of Runx2, and Akt signaling is upstream of CypA.

Naphthenic acid fraction components from oil sands process-affected water from the Athabasca Oil Sands Region impair murine osteoblast differentiation and function.

The extraction of bitumen from surface mining in the Athabasca Oil Sands Region (AOSR) produces large quantities of oil sands process-affected water (OSPW) that needs to be stored in settling basins near extraction sites. Chemical constituents of OSPW are known to impair bone health in some organisms, which can lead to increased fracture risk and lower reproductive fitness. Naphthenic acid fraction components (NAFCs) are thought to be among the most toxic class of compounds in OSPW; however, the effect of NAFCs on osteoblast development is largely unknown. In this study, we demonstrate that NAFCs from OSPW inhibit osteoblast differentiation and deposition of extracellular matrix, which is required for bone formation. Extracellular matrix deposition was inhibited in osteoblasts exposed to 12.5-125 mg/L of NAFC for 21 days. We also show that components within NAFCs inhibit the expression of gene markers of osteoblast differentiation and function, namely, alkaline phosphatase (Alp), osteocalcin, and collagen type 1 alpha 1 (Col1a1). These effects were partially mediated by the induction of glucocorticoid receptor (GR) activity; NAFC induces the expression of the GR activity marker genes Sgk1 (12.5mg/L) and p85a (125 mg/L) and inhibits GR protein (125 mg/L) and Opg RNA (12.5mg/L) expression. This study provides evidence that NAFC concentrations of 12.5mg/L and above can directly act on osteoblasts to inhibit bone formation and suggests that NAFCs contain components that can act as GR agonists, which may have further endocrine disrupting effects on exposed wildlife.

FOXO1 differentially regulates bone formation in young and aged mice

It is a great challenge to develop a safe and effective treatment strategy for age-related osteoporosis and fracture healing. As one of the four FOXO transcription factors, FOXO1 is essential for cell proliferation, survival, senescence, energy metabolism, and oxidative stress in various cells. Our previous study demonstrated that specific Foxo1 gene deletion in osteoblasts in young mice results in bone loss while that in aged mice shows the opposite effect. However, the mechanism underlying the differential regulation of bone metabolism by FOXO1 remains to be elucidated. In this study, we generated osteoblast-specific Foxo1 knockout mice by using Foxo1fl/fl and Bglap-Cre mice. In young mice, Foxo1 gene deletion inhibits osteoblast differentiation, leading to a decreased osteoblast number and decreased bone formation rate because of the weakened ability to resist oxidative stress, eventually resulting in bone loss and delayed healing of bone defects. In aged mice, high levels of reactive oxygen species (ROS) promote the diversion of CTNNB1 (β-catenin) from T cell factor 4 (TCF4)- to FOXO1-mediated transcription, thereby inhibiting Wnt/β-catenin signaling and leading to decreased osteogenic activity. Conversely, FOXO1 deficiency indirectly promotes the binding of β-catenin and TCF4 and activates Wnt/β-catenin signaling, thereby alleviating age-related bone loss and improving bone defect healing. Our study proves that FOXO1 has differential effects on bone metabolism in young and aged mice and elucidates its underlying mechanism. Further, this study provides a new perspective on the treatment of age-related osteoporosis.