Decreased Alkaline Phosphatase Activity Research Articles

MiR-183 regulates the differentiation of osteoblasts in the development of osteoporosis by targeting Smad4

ObjectiveTo discuss the effect of miR-183 on osteoblast differentiation in the osteoporosis progression via targeting Smad4. MethodsOsteoporosis models were constructed on ovariectomized (OVX) mice to determine the expression of miR-183 and Smad4. Then, MC3T3-E1 cells and primary osteoblasts were divided into Mock, miR-control, miR-183 mimic, miR-183 inhibitor, siSmad4 and miR-183 inhibitor + siSmad4 groups. Alkaline phosphatase (ALP) staining were performed to determine ALP activity, alizarin red staining to evaluate the calcium deposit, while qRT-PCR and Western blotting were used to determine the expression of related molecules. Besides, MC3T3-E1 cells transfected with miR-control or miR-183 mimic were cultured with or without TGF-β1 to verify whether miR-183 regulates the TGF-β signaling pathway. ResultsMiR-183 was up-regulated with decreased Smad4 in the femur of OVX mice, and dual luciferase reporter gene assay showed that Smad4 was a target of miR-183. As compared to Mock group, MC3T3-E1 cells and primary osteoblasts in the miR-183 mimic group and siSmad4 group had significant reductions of OCN, OPN, Runx2 and Osx, as well as decreased ALP activity and calcium deposit. Contrarily, miR-183 and Smad4 were up-regulated and down-regulated respectively. However, cells in the miR-183 inhibitor group manifested the opposite changes. Besides, osteoblast differentiation in the miR-183 inhibitor + siSmad4 group was weakened evidently when compared to miR-183 inhibitor group. Pathway analysis indicated that miR-183 regulated osteogenic differentiation via TGF-β signaling pathway. ConclusionMiR-183 was up-regulated in osteoporosis, and miR-183 overexpression can inhibit osteoblast differentiation by targetedly down-regulating TGF-β pathway member Smad4 to trigger osteoporosis.

Toxicity evaluation and oxidative stress response of fumaronitrile, a persistent organic pollutant (POP) of industrial waste water on tilapia fish (Oreochromis mossambicus)

The study was designed to determine the impact of acute toxicity of fumaronitrile exposure through tissue damaging, oxidative stress enzymes and histopathological studies in gills, liver and muscle cells of freshwater tilapia fish (Oreochromis mossambicus). In gill, liver, and muscle cells, biochemical indicators such as tissue damage enzymes (Acid Phosphatase (ACP), Alkaline Phosphatase (ALP), and Lactate Dehydrogenase (LDH)) and antioxidative enzymes (Superoxide Dismutase (SOD); Catalase (CAT); Glutathione-S-transferase (GST); Reduced Glutathione (GSH); Glutamate oxaloacetate transaminase (GOT) and Glutamate pyruvate transaminase (GPT) were quantified in the time interval of 30, 60 and 90 days exposure to the fumaronitrile. After 90 days, under 6 ppb exposure conditions, the acid phosphatase (ACP) levels of fish increased significantly in the gills (3.439 μmol/mg protein/min), liver (1.743 μmol/mg protein/min), and muscles (2.158 μmol/mg protein/min). After 90 days of exposure to the same concentration and days, ALP activity increased significantly in gills (4.354 μmol/mg protein/min) and liver (1.754 μmol/mg protein/min), but muscle cells had a little decrease in ALP activity (2.158 μmol/mg protein/min). The LDH concentration in gills following treatment with fumaronitrile over a period of 0–90 days was 3.573 > 3.521 > 2.245 μmol/mg protein/min over 30 > 60 > 90 days. However, at the same dose and treatment duration, a greater LDH level of 0.499 μmol/mg protein/min was found in liver and muscle cells. Histopathological abnormalities in the gills, liver, and muscle cells of treated fish were also examined, indicating that fumaronitrile treatment generated the most severe histological changes. The current study reveals that fumaronitrile exposure has an effect on Oreochromis mossambicus survival, explaining and emphasising the risk associated with this POP exposure to ecosystems and human populations.

Interactions between cadmium and zinc on gene expression pattern of differentiation markers in MC3T3-E1 cell line

We evaluated, in vitro, the interactions between cadmium (Cd) and zinc (Zn) during the proliferation and differentiation process using bone MC3T3-E1 cell line. Cells were treated with CdCl2 and/or ZnCl2 for 24 and 48 h and 5 µM CdCl2 was found as low cytotoxic dose and 25 µM ZnCl2 as the best Zn treatment for cell proliferation. Gene expression of some bone markers (Runx2, collagen α1 (Colα1), osteocalcin (Oc), alkaline phosphatase (ALP) and bone sialoprotein (BSP)) was studied at 24, 48 and 72 h. Treatment by CdCl2 depressed Runx2, Colα1, and BSP mRNA levels after 24 h. Oc and ALP gene expression was found to be decreased after 72 h. CdCl2 -exposure decreased ALP activity and Ca deposit in matrix. In concomitant treatment by CdCl2 and ZnCl2, gene expression of osteoblastic markers was found to be up-regulated (p < 0, 05) compared to CdCl2 treated cells, ALP staining and mineralization were increased. Our results show that Zn could prevent Cd-induced toxicity on MC3T3-E1 cells, probably through the restoration of Runx2, col α1, BSP, ALP and Oc and gene expression inhibited by Cd.

The role of long noncoding RNA THAP9-AS1 in the osteogenic differentiation of dental pulp stem cells via the miR-652-3p/VEGFA axis.

Dental pulp stem cells (DPSCs) are multipotent and may play crucial roles in dentin-pulp regeneration. Recent studies have revealed that long noncoding RNAs (lncRNAs) are implicated in the osteogenic differentiation of DPSCs. However, the specific role and potential mechanisms of the lncRNA trihydroxyacetophenone domain containing nine antisense RNA 1 (THAP9-AS1) during osteogenic differentiation of DPSCs remain unknown. In the present study, we determined that THAP9-AS1 expression was upregulated during osteogenic differentiation of DPSCs. Moreover, we investigated the biological functions of THAP9-AS1 during osteogenic differentiation of DPSCs by loss-of-function assays. THAP9-AS1 knockdown inhibited osteogenic differentiation of DPSCs by decreasing alkaline phosphatase activity, alkaline phosphatase-positive cell ratio, mineralizing matrix and mRNA, and protein levels of early osteogenic-markers. We also found that THAP9-AS1 interacted with miR-652-3p, whose downstream gene target is vascular endothelial growth factor A (VEGFA). In addition, rescue assays indicated that VEGFA rescued the effects of THAP9-AS1 knockdown during osteogenic differentiation of DPSCs. In summary, we verified that knockdown of THAP9-AS1 inhibits osteogenic differentiation of DPSCs via the miR-652-3p/VEGFA axis. Our findings may be helpful to extend research on the mechanisms underlying osteogenic differentiation of DPSCs.

Hyperglycemia minimally alters primary self-renewing human colonic epithelial cells while TNFα-promotes severe intestinal epithelial dysfunction.

Hyperglycemia is thought to increase production of inflammatory cytokines and permeability of the large intestine. Resulting intestinal inflammation is then often characterized by excess secretion of tumor necrosis factor alpha (TNFα). Thus, hyperglycemia in hospitalized patients suffering from severe trauma or disease is frequently accompanied by TNFα secretion, and the combined impact of these insults on the intestinal epithelium is poorly understood. This study utilized a simple yet elegant model of the intestinal epithelium, comprised of primary human intestinal stem cells and their differentiated progeny, to investigate the impact of hyperglycemia and inflammatory factors on the colonic epithelium. When compared to epithelium cultured under conditions of physiologic glucose, cells under hyperglycemic conditions displayed decreased mucin-2 (MUC2), as well as diminished alkaline phosphatase (ALP) activity. Conditions of 60mM glucose potentiated secretion of the cytokine IL-8 suggesting that cytokine secretion during hyperglycemia may be a source of tissue inflammation. TNFα measurably increased secretion of IL-8 and IL-1β, which was enhanced at 60mM glucose. Surprisingly, intestinal permeability and paracellular transport were not altered by even extreme levels of hyperglycemia. The presence of TNFα increased MUC2 presence, decreased ALP activity, and negatively impacted monolayer barrier function. When TNFα hyperglycemia and≤30mM glucose and were combined, MUC2 and ALP activity remained similar to that of TNFα alone, although synergistic effects were seen at 60mM glucose. An automated image analysis pipeline was developed to assay changes in properties of the zonula occludens-1 (ZO-1)-demarcated cell boundaries. While hyperglycemia alone had little impact on cell shape and size, cell morphologic properties were extraordinarily sensitive to soluble TNFα. These results suggest that TNFα acted as the dominant modulator of the epithelium relative to glucose, and that control of inflammation rather than glucose may be key to maintaining intestinal homeostasis.

The Effects of MiR-320 on the Proliferation and Differentiation of Human Alveolar Bone-Derived Mesenchymal Stem Cells

Alveolar bone-derived mesenchymal stem cells (AB-BMSCs) have a biological morphology and antigen phenotype similar to those of BMSCs. However, the intrinsic characteristics of AB-BMSCs and their underlying mechanisms, in which the involvement of micro(mi)RNAs has been reported, remain unknown. This study shows that miR-320c expression was significantly suppressed during osteoblastic differentiation of human AB-BMSCs. The overexpression of miR-320c markedly decreased cellular proliferation, intracellular activity of alkaline phosphatase (ALP) and formation of calcium nodules; mRNA levels of osteogenesis-related genes were significantly reduced compared to those in control cells. Calcium nodule formation in miR-320c-knockdown cells was significantly increased, andHOXA10, Runx2,andBGPmRNA levels were significantly increased compared to those in control cells. These results indicate that miR-320c suppresss the proliferation and osteogenic differentiation of AB-BMSCs, in part by decreasing ALP activity, cellular proliferation, mineralization, and expression of several osteogenesis-related genes. These results lay the basic foundation for the elucidation of the molecular mechanisms of alveolar bone reconstruction.

Nano-Sized Hydroxyapatite Induces Apoptosis and Osteogenic Differentiation of Vascular Smooth Muscle Cells via JNK/c-JUN Pathway.

PurposeThe deposition of hydroxyapatite (HAp) crystals plays an important role in the development of vascular calcification (VC). This study aimed to demonstrate the effects of nanosized HAp (nHAp) on vascular smooth muscle cells (VSMCs) and VC progression.MethodsTransmission electron microscopy (TEM) was used to examine cellular uptake of nHAp. Cell viability was determined using CCK-8 assay kit. Mitochondrial impairment and reactive oxygen species were detected by TEM and fluorescence dye staining, respectively. Cell apoptosis was detected by Western blot analysis and Annexin V staining. Mouse model of VC was built via applying nHAp on the surface of abdominal aorta. Calcification was visualized by Alizarin red and von Kossa staining.ResultsWe found that nHAp could promote osteogenic transformation of VSMCs by elevating expression of runt-related factor 2 (Runx2), osteopontin (OPN) and alkaline phosphatase (ALP), impairing function and morphology of mitochondria and inducing apoptosis of VSMCs. More phosphorylation of c-Jun N-terminal protein kinase/c-JUN (JNK/c-JUN) in VSMCs was detected after mixing nHAp with VSMCs. HAp-induced osteogenic transformation of VSMCs was blocked by JNK inhibitor SP600125, resulted in decreased ALP activity, less Runx2 and OPN expressions. SP600125 also inhibited apoptosis of VSMCs. Application of nHAp to outside of aorta induced osteogenic transformation and apoptosis of VSMCs, and significant deposition of calcium on the vessel walls of mice, which can be effectively attenuated by SP600125.ConclusionJNK/c-JUN signaling pathway is critical for nHAp-induced calcification, which could be a potential therapeutic target for controlling the progression of VC.

Endothelial to Mesenchymal Transformation-derived Activated Fibroblast Behavior in a 3D Culture Environment

Objective: In calcific aortic valve disease (CAVD), glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) and hyaluronic acid (HA) are present in the normally collagen-rich aortic valve fibrosa near calcified nodules. Previous work using a 3D in vitro model has shown that GAGs can also promote endothelial to mesenchymal transformation (EndMT). The role of mesenchymal transformation in the progression of CAVD remains unclear. The goal of this study is to investigate the role of GAGs and EndMT on valve cell behavior in a 3D, in vitro model of healthy and diseased valves. Methods: Three dimensional in-vitro cultures, representing healthy (collagen 1 hydrogels) or diseased (collagen 1 hydrogels + GAGs) valve tissue, were seeded with Porcine Aortic Valve Endothelial Cells (PAVEC) and Porcine Aortic Valve Interstitial Cells (PAVIC). Cell cultures were grown for 14 days in regular medium and then evaluated for calcified nodule formation, alkaline phosphatase activity, proliferation and protein expression relating to EndMT activity Results: Introduction of GAGs into the in vitro model resulted in decreased alkaline phosphatase activity. Hydrogels containing CS conditions induced the greatest calcification, as measured with Alzarin Red quantification, while gels containing HA conditions did not have calcification that was significantly different than controls. Proliferation results suggest high levels of cell division activity, for both PAVEC and PAVIC in CS conditions. Protein expression analysis indicated increased EndMT in hydrogels containing GAGs. Conclusions: 3D, in vitro models of the aortic valve simulated progression toward late stage CAVD. Further investigation of the downstream role of EndMT within the aortic valve microenvironment is critical to subsequent improvement in treatment options.

Chemically and Green Synthesized ZnO Nanoparticles Alter Key Immunological Molecules in Common Carp (Cyprinus carpio) Skin Mucus.

This study was conducted to compare the effects of commercially available (C) and green synthesized (GS) Zinc oxide nanoparticles (ZnO-NPs) on immunological responses of common carp (Cyprinus carpio) skin mucus. GS ZnO-NPs were generated using Thymus pubescent and characterized by UV–vis diffuse reflectance spectroscopy (DRS), Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), scanning electron microscope (SEM), and energy-dispersive X-ray spectroscopy (EDX). Fish (n = 150) were randomly allocated into five groups in triplicate and received a waterborne concentration of 0% (control), 25%, and 50% of LC50 96 h of commercially available (C1 and C2) and green synthesized ZnO-NPs (GS1 and GS2) for 21 days. Results from XRD displayed ZnO-NPs with 58 nm in size and UV-vis DRS, EDX, and FT-IR analysis showed that some functional groups from plant extract bonded to the surface of NPs. The SEM images showed that ZnO-NPs have conical morphology. Acute toxicity study showed a higher dose of LC5096h for green synthesized ZnO-NPs (78.9 mg.L−1) compared to the commercial source (59.95 mg.L−1). The highest activity of lysozyme and alternative complement activity (ACH50) were found in control and GS1 groups. A significant decrease in alkaline phosphatase activity (ALP) was found in C1 and C2 groups compared to other treatments. Protease activity (P) was significantly decreased in the C2 group compared to the control and GS groups. Total immunoglobulin (total Ig) content was the highest in the control. In addition, total Ig in the GS1 group was higher than GS2. The exposure to ZnO-NPs lowered total protein content in all experimental groups when compared to control. Present findings revealed lower induced immunosuppressive effects by green synthesized ZnO-NPs on key parameters of fish skin mucus.

Interleukin-20 inhibits the osteogenic differentiation of MC3T3-E1 cells via the GSK3β/β-catenin signalling pathway

ObjectiveTo investigate the effects of interleukin-20 (IL-20) on the osteogenic differentiation of MC3T3-E1 cells. MethodsThe pre-osteoblast line MC3T3-E1 was treated with different concentrations of IL-20 (0, 2, 20 and 100 ng/mL), and the cell viability was detected by the CCK8 assay. To assess the influence of IL-20 on osteogenic differentiation, alkaline phosphatase (ALP) activity and Alizarin red staining were performed at predetermined times. The expression levels of Runt-related transcription factor 2 (RUNX2), Osterix (Osx), glycogen synthase kinase-3β (GSK-3β) and β-catenin were detected by qRT-PCR and Western blotting analyses. 5 nmol/L lithium chloride (LiCl) was used as GSK-3β inhibitor. ResultsIL-20 promoted cell proliferation but decreased ALP activity and mineralization. Moreover, IL-20 downregulated the expression of RUNX2, Osx and β-catenin but upregulated the level of GSK-3β. ConclusionsThe results suggest that IL-20 could inhibit the osteogenic differentiation of MC3T3-E1 cells via the GSK3β/β-catenin signalling pathway.

Optimizing In Vitro Osteogenesis in Canine Autologous and Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells with Dexamethasone and BMP-2.

A growing body of work suggests that canine mesenchymal stromal cells (cMSCs) require additional agonists such as bone morphogenic protein-2 (BMP-2) for consistent in vitro osteogenic differentiation. BMP-2 is costly and may challenge the translational relevance of the canine model. Dexamethasone enhances osteogenic differentiation of human MSCs (hMSCs) and is widely utilized in osteogenic protocols. The aim of this study was to determine the effect of BMP-2 and dexamethasone on early- and late-stage osteogenesis of autologous and induced pluripotent stem cell (iPS)-derived cMSCs. Two preparations of marrow-derived cMSCs were selected to represent exceptionally or marginally osteogenic autologous cMSCs. iPS-derived cMSCs were generated from canine fibroblasts. All preparations were evaluated using alkaline phosphatase (ALP) activity, Alizarin Red staining of osteogenic monolayers, and quantitative polymerase chain reaction. Data were reported as mean ± standard deviation and compared using one- or two-way analysis of variance and Tukey or Sidak post hoc tests. Significance was established at P < 0.05. In early-stage assays, dexamethasone decreased ALP activity for all cMSCs in the presence of BMP-2. In late-stage assays, inclusion of dexamethasone and BMP-2 at Day 1 of culture produced robust monolayer mineralization for autologous cMSCs. Delivering 100 nM dexamethasone at Day 1 improved mineralization and reduced the BMP-2 concentrations required to achieve mineralization of the marginal cMSCs. For iPS-cMSCs, dexamethasone was inhibitory to both ALP activity and monolayer mineralization. There was increased expression of osteocalcin and osterix with BMP-2 in autologous cMSCs but a more modest expression occurred in iPS cMSCs. While autologous and iPS-derived cMSCs respond similarly in early-stage osteogenic assays, they exhibit unique responses to dexamethasone and BMP-2 in late-stage mineralization assays. This study demonstrates that dexamethasone and BMP-2 can be titrated in a time- and concentration-dependent manner to enhance osteogenesis of autologous cMSC preparations. These results will prove useful for investigators performing translational studies with cMSCs while providing insight into iPS-derived cMSC osteogenesis.

Effects of Advanced Glycation End Products on Differentiation and Function of Osteoblasts and Osteoclasts.

BackgroundRisk of fragility fractures increases in patients with diabetes mellitus, independent of bone mineral density. In the present study, the effects of advanced glycation end products (AGEs) on differentiation and function of osteoblasts and osteoclasts were investigated.MethodsAGEs and 25 mM glucose were administered to marrow-derived macrophages and MCT3T3-E1 cells. The effects of AGEs on osteoclast differentiation was investigated using tartrate-resistant acid phosphatase (TRAP) assay. The effects of AGEs on osteoblast differentiation was investigated using alkaline phosphatase (ALP) activity and bone nodule formation assays. Expression of osteoclast-specific and osteoblast-specific genes and effects on cell signaling pathways associated with cell differentiation were analyzed using reverse transcription polymerase chain reaction and western blotting.ResultsAGEs significantly decreased TRAP-positive multinucleated cell formation in receptor activator of nuclear factor-κB ligand-induced marrow-derived macrophages in a dose-dependent manner. AGEs suppressed the expression of osteoclast-specific genes, JNK, p38, AKT, intercellular adhesion molecule 1, and lymphocyte function-associated antigen 1 in marrow-derived macrophages. AGEs decreased ALP activity and showed a tendency to decrease bone nodule formation in MC3T3-E1 cells. AGEs suppressed the expression of osteoblast-specific genes, lysyl hydroxylase and lysyl oxidase in MC3T3-E1 cells.ConclusionAGEs suppressed differentiation and function of osteoclasts and osteoblasts, and collagen cross-linking activity. It suggests that AGE may induce bone fragility through low bone turnover and deterioration of bone quality.

Ubiquitin specific protease 7 maintains pluripotency of mouse embryonic stem cells through stabilization of β-catenin.

Embryonic stem cells (ESCs), which are derived from the undifferentiated inner cell mass of the embryo, can differentiate every cell type of the body regarding their pluripotency. Therefore, human or mouse ESCs can be used as an unlimited cell source for numerous researches or therapeutical approaches. However, pluripotency maintenance of ESCs during in vitro culture is challenging because of their endless differentiation capacity. In the current study, the effect of USP7 on pluripotency maintenance of mouse ESCs (mESCs) has been investigated with the help of cell viability assay, morphological analysis, alkaline phosphatase (ALP) staining, qPCR analysis, and Western Blotting. 600 nM P5091 application, which showed no significant toxicity in mESCs, increased the total ubiquitinated protein amount as a proof of the accomplishment of proper USP7 inhibition. Morphological analysis and ALP activity evaluation indicated that dual inhibition of GSK3 and MEK together with leukemia inhibitory factor (LIF) treatment protects the pluripotency in presence of active USP7 enzyme. Yet, inactivation of USP7 reduced the ALP activity and altered the cell morphology in each treatment group. This morphological change and decreased ALP activity refer to differentiated mESCs. These findings were supported by gene expression and protein analysis. Gene expressions and protein amounts of pluripotency related Oct4, Nanog, c-Myc, Sox2 and Klf4 transcription factors decreased significantly after USP7 inhibition. Together with this observation, a remarkable reduction in β-Catenin expression was also noticed. It was also observed that USP7 inactivation shortens the half-live of β-Catenin and GSK3β proteins. This study demonstrates that USP7 activation is crucial for proper pluripotency maintenance, which is provided through β-Catenin stabilization.

In situ electrochemical analysis of alkaline phosphatase activity in 3D cell cultures

In bioelectrochemistry, cells are typically cultured in monolayers on 2D electrodes and methods such as electrochemical impedance spectroscopy (EIS) or voltammetry are used to detect changes in the cell population or responses to external stimuli. Here, we implement an electrochemical assay for the in situ monitoring of alkaline phosphatase (ALP) activity of Saos-2 cells cultured in a 3D gelatin hydrogel matrix with a thickness of 300 µm deposited on pyrolytic carbon electrodes. After cell seeding, ALP activity for cells in the hydrogel was higher compared to 2D cell cultures probably due to unrestricted access to the enzyme on the complete cell membrane. Gradual decrease of cell proliferation, maturation and spheroid formation in the 3D cell culture resulted in slower increase and eventually a decrease in ALP activity over time. Finally, 3D microstructured electrodes and 3D carbon pillar microelectrodes with a height of 225 µm were fabricated and applied for ALP detection directly in the 3D cell cultures. This resulted in higher electrochemical signals compared to 2D electrode configurations due to increased electrode surface area and penetration of the microelectrodes into the cell-laden hydrogel.

MicroRNA‑204‑5p inhibits the osteogenic differentiation of ankylosing spondylitis fibroblasts by regulating the Notch2 signaling pathway.

Ankylosing spondylitis (AS) is a chronic inflammatory systemic disease and is difficult to detect in the early stages. The present study aimed to investigate the role of microRNA (miR)-204-5p in osteogenic differentiation of AS fibroblasts. Bone morphogenetic protein 2 (BMP-2) was used to induce osteogenic differentiation. Cells were divided into the following groups: AS group, AS + BMP-2 group, AS + BMP-2 + miR-negative control group, AS + BMP-2 + miR-204-5p mimics group and AS + BMP-2 + miR-204-5p mimics + pcDNA-Notch2 group. The expression levels of miR-204-5p, Notch2, runt-related transcription factor 2 (RUNX2) and osteocalcin were detected via reverse transcription-quantitative PCR analysis. The binding site between Notch2 and miR-204-5p was predicted using TargetScan software and verified via the dual-luciferase reporter assay. Alkaline phosphatase (ALP) activity was assessed via the ALP assay, while the mineralized nodules area was determined via the Alizarin Red S staining assay. The results demonstrated that Notch2 is a target gene of miR-204-5p. Furthermore, treatment with BMP-2 significantly decreased miR-204-5p expression, and significantly increased ALP activity, the mineralized nodules area and the expression levels of Notch2, RUNX2 and osteocalcin in ligament fibroblasts (all P<0.05). Conversely, transfection with miR-204-5p mimics significantly increased miR-204-5p expression, and significantly decreased ALP activity, the mineralized nodules area and the expression levels of Notch2, RUNX2 and osteocalcin in ligament fibroblasts (all P<0.05). Notably, transfection with pcDNA-Notch2 significantly reversed the inhibitory effects induced by miR-204-5p mimics on the osteogenic differentiation of ligament fibroblasts (all P<0.05). Furthermore, miR-204-5p inhibited the osteogenic differentiation of ligament fibroblasts in patients with AS by targeting Notch2. Thus, miR-204-5p may negatively regulate Notch2 expression and may be a potential therapeutic target for AS. Collectively, the results of the present study provide a theoretical basis for the effective treatment of patients with AS.

A validated miRNA signature for the diagnosis of osteoporosis related fractures using SVM algorithm classification.

The aim of the present study was to develop a circulating microRNA expression signature for early prediction of osteoporotic fractures and to validate the results using Gene Expression Omnibus (GEO) datasets. The GSE70318 dataset was downloaded from GEO and used to build an osteoporotic fracture prediction model based on the receiver operating characteristic curve and support vector machine (SVM) classification index. The GSE74209 dataset was used as a validation dataset. Additionally, in vitro, alkaline phosphatase (ALP) activity was measured in the presence or absence of microRNA (miRNA/miR) treatments in human osteoblast cells. The expression of two selected genes was detected by western blotting. miR-188-3p, miR-942-3p, miR-576-3p and miR-135a-5p were differentially expressed between controls and osteoporotic patients with fractures. SVM classification using these four miRNAs provided better dichotomization. It was further confirmed that miR-576-3p and 135a-5p in the GSE74209 dataset could also significantly discriminate between the controls and fracture patients, the area under the curve of SVM2 was 0.9722 with 95% CI 0.8885-1.056. Further analysis indicated that the target genes of the two miRNAs participated in the Wingless-related integration site, Hedgehog and transforming growth factor-β signaling pathways and osteoclast differentiation. miR-576-3p and miR-135-5p transfection decreased ALP activity and ALP activity was increased in the presence of blocking antisense oligonucleotides. Western blotting indicated miR-576-3p and miR-135-5p decreased CSNK1A1L and LRP6 levels, respectively. In conclusion, two miRNA signatures were developed and validated for the prediction of osteoporotic fractures.

PERK-eIF2\u03b1-ATF4 signaling contributes to osteogenic differentiation of periodontal ligament stem cells

Protein kinase-like endoplasmic reticulum kinase (PERK) is a type I transmembrane protein located in the endoplasmic reticulum (ER). The PERK-eukaryotic initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway has been proved to be involved in osteoblast differentiation, but the involvement of the PERK-eIF2α-ATF4 signaling pathway in osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) has remained unclear. Therefore, the aim of this study was to explore the role of PERK in osteogenic differentiation of hPDLSCs and to assess whether PERK-eIF2α-ATF4 contributes to the process of osteogenic differentiation in hPDLSCs. In our study, we constructed PERK-overexpressed and PERK-silenced hPDLSCs by lentiviral transduction. Furthermore, lentivirus-transfected cells were induced to differentiate into osteoblast cells for different days. Alkaline phosphatase (ALP) activity and Alizarin Red staining were used to evaluate the mineralization capacity, and the expression levels of related genes-ATF4, ALP, bone sialoprotein, runt-related transcription factor 2 (Runx2), and osteocalcin were measured to evaluate the osteogenic differentiation of hPDLSCs. The results showed that over-expression of PERK greatly increased ALP activity and the expression levels of related osteogenic genes, which displayed the strongest osteogenesis capacity. However, suppression of PERK caused decreased ALP activity and the weakest osteogenesis capacity, and the levels of ATF4 and p-eIF2α in PERK-silenced hPDLSCs were also decreased. Our results indicated that the PERK gene plays an important role in the differentiation of hPDLSCs to osteoblast-like cells. The PERK-eIF2α-ATF4 signaling pathway contributes to osteoblast differentiation of hPDLSCs.

Catalpol Promotes the Proliferation and Differentiation of Osteoblasts Induced by High Glucose by Inhibiting KDM7A.

IntroductionThe protective effect of catalpol on diabetic osteoporosis (DOP) and its mechanism remain unclear. This study aimed to explore whether catalpol enhanced the proliferation and differentiation of MC3T3 cells induced by high glucose by inhibiting the expression of KDM7A.MethodsMC3T3 cells were induced by high glucose (HG) and treated with different concentrations of catalpol. The proliferation and mineralization abilities of MC3T3-E1 cells were determined by CCK-8 assay and Alizarin Red Staining, respectively. The expression of differentiation-related osteogenic proteins, KDM7A and related proteins of Wnt/β-catenin signaling pathway was analyzed by Western blot analysis. The alkaline phosphatase (ALP) activity was detected by ALP assay kits.ResultsMC3T3-E1 cells induced by high glucose showed decreased proliferation and mineralization abilities and decreased ALP activity, which were all reversed by the treatment of catalpol. High glucose induction inhibited the expression of KDM7A, Total-β-catenin, Nuclear-β-catenin and p-GSK3β, which was reversed by the treatment of catalpol. And KDM7A interference up-regulated the expression of Total-β-catenin, Nuclear-β-catenin and p-GSK3β, which was down-regulated by KDM7A overexpression. Furthermore, the proliferation and mineralization abilities and ALP activity were improved when treated with KDM7A interference and decreased when treated with KDM7A overexpression. However, SKL2001 could improve the proliferation and mineralization abilities and ALP activity of MC3T3-E1 cells.DiscussionCatalpol promotes the proliferation and differentiation of osteoblasts induced by high glucose by regulating the Wnt/β-catenin signaling pathway through KDM7A.

Transforming Growth Factor Beta (TGF-β) Regulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Promoting Wnt Signaling Pathway in Atrophic Nonunion

During atrophic nonunion, Wnt signaling pathway is inhibited, resulting in inhibition of BMSC osteogenic differentiation. TGF-β regulates growth and development of the body. However, TGF-β’s effect on osteogenic differentiation of BMSCs in atrophic nonunion has not been reported. The bone tissue and serum of patients with atrophic nonunion and normal healing fractures were collected, and TGF-β mRNA and serum secretion were analyzed by Real time PCR and ELISA. Rat BMSCs were cultured and randomly divided into control group, TGF-β group and TGF-β siRNA group which was transfected with pcDNA-TGF-β plasmid or TGF-β siRNA respectively followed by analysis of cell proliferation by MTT assay, alkaline phosphatase (ALP) activity, Caspase3 activity, expression of RUNX2 and OPN and PPARγ2 mRNA by Real time PCR, and WNT5A and FZD3 expression by Western blot. TGF-β mRNA level and secretion in patients with atrophic nonunion was significantly reduced compared with patients with normal healing fractures (P &lt; 0.05). Transfection of TGF-β siRNA down-regulated TGF-β expression in BMSCs, significantly inhibited cell proliferation, increased Caspase3 activity, decreased ALP activity, RUNX2 and OPN expression, increased PPARγ2 expression and deceased WNT5A and FZD3 expression (P &lt; 0.05). However, transfection of pcDNA-TGF-β plasmid up-regulated TGF-β expression in BMSCs and reversed the above changes (P &lt; 0.05). TGF-β is reduced in atrophic nonunion patients. Targeting TGF-β promotes BMSCs proliferation and osteogenic differentiation by regulating Wnt signaling pathway.

Effect of Hepatic Kinase B1 (LKB1) on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells During Senescence

Hepatic kinase B1 (LKB1) is a tumor suppressor and regulates cell proliferation and apoptosis. However, whether LKB1 affects bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation of during aging remains unclear. Two BMSCs derived from Zempster24−/− (aging) and Zempster24+/+ (normal) mice were cultured in vitro followed by measurement of LKB1 expression by real-time quantitative PCR and Western blot. LKB1 siRNA was transfected into Zempster24−/−BMSCs and LKB1 expression was measured. 14 days after osteogenic induction, mineralized nodule formation was evaluated by alizarin red staining, expression of Calcin, type I collagen, RUNX2 and OPN mRNA expression was measured, together with alkaline phosphatase (ALP) activity and the PI3K/mTOR pathway activity. Compared with normal BMSCs, LKB1 expression was significantly increased, calcified nodules were decreased, with reduced expression of osteocalcin, type I collagen, RUNX2 and OPN mRNA as well as decreased ALP activity and PI3K/mTOR signaling protein expression (P &lt; 0.05). LKB1 siRNA transfection into senescent BMSCs down-regulated LKB1 expression, increased calcification nodule formation, expression of osteocalcin, type I collagen, RUNX2 and OPN mRNA, as well as increased ALP activity and PI3K/mTOR pathway protein expression (P &lt; 0.05). Aging can promote the increase of LKB1 expression and inhibit the osteogenic differentiation of BMSCs. Down-regulation of LKB1 expression in BMSCs during senescence can promote osteogenic differentiation through regulating PI3K/mTOR pathway.