scholarly journals Quantification of Gene Expression with a Secreted Alkaline Phosphatase Reporter System

BioTechniques ◽  
1997 ◽  
Vol 23 (6) ◽  
pp. 1110-1114 ◽  
Author(s):  
Te-Tuan Yang ◽  
Parisa Sinai ◽  
Paul A. Kitts ◽  
Steven R. Kain
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Reporter System

scholarly journals Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway

2015 ◽  
Vol 112 (41) ◽  
pp. 12711-12716 ◽  
Author(s):  
Andrea M. Brum ◽  
Jeroen van de Peppel ◽  
Cindy S. van der Leije ◽  
Marijke Schreuders-Koedam ◽  
Marco Eijken ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Osteoblast Differentiation ◽  
Target Genes ◽  
Focal Adhesions ◽  
Bone Fragility ◽  
Bone Morphogenetic Protein 2 ◽  
Marker Genes ◽  
Connectivity Map ◽  
Morphogenetic Protein

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL, osteopontin/SPP1, and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity.

scholarly journals pTRA – A reporter system for monitoring the intracellular dynamics of gene expression

PLoS ONE ◽  
2018 ◽  
Vol 13 (5) ◽  
pp. e0197420 ◽  
Author(s):  
Sabine G. Wagner ◽  
Martin Ziegler ◽  
Hannes Löwe ◽  
Andreas Kremling ◽  
Katharina Pflüger-Grau
Keyword(s):  
Gene Expression ◽  
Reporter System

The influence of titanium surfaces treated by alkalis on macrophage and osteoblast-like cell adhesion and gene expression in vitro

RSC Advances ◽  
2015 ◽  
Vol 5 (99) ◽  
pp. 81378-81387 ◽  
Author(s):  
Ting Ma ◽  
Xi-Yuan Ge ◽  
Sheng-Nan Jia ◽  
Xi Jiang ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Cell Adhesion ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Related Gene ◽  
Titanium Surfaces

The effect of alkali-treated titanium surfaces on inflammation-related gene expression of macrophages and alkaline phosphatase activity of osteoblast-like cells.

Simulated microgravity using the Random Positioning Machine inhibits differentiation and alters gene expression profiles of 2T3 preosteoblasts

2005 ◽  
Vol 288 (6) ◽  
pp. C1211-C1221 ◽  
Author(s):  
Steven J. Pardo ◽  
Mamta J. Patel ◽  
Michelle C. Sykes ◽  
Manu O. Platt ◽  
Nolan L. Boyd ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Bone Loss ◽  
Simulated Microgravity ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Underlying Mechanism ◽  
Bone Biology ◽  
Random Positioning Machine

Exposure to microgravity causes bone loss in humans, and the underlying mechanism is thought to be at least partially due to a decrease in bone formation by osteoblasts. In the present study, we examined the hypothesis that microgravity changes osteoblast gene expression profiles, resulting in bone loss. For this study, we developed an in vitro system that simulates microgravity using the Random Positioning Machine (RPM) to study the effects of microgravity on 2T3 preosteoblast cells grown in gas-permeable culture disks. Exposure of 2T3 cells to simulated microgravity using the RPM for up to 9 days significantly inhibited alkaline phosphatase activity, recapitulating a bone loss response that occurs in real microgravity conditions without altering cell proliferation and shape. Next, we performed DNA microarray analysis to determine the gene expression profile of 2T3 cells exposed to 3 days of simulated microgravity. Among 10,000 genes examined using the microarray, 88 were downregulated and 52 were upregulated significantly more than twofold using simulated microgravity compared with the static 1-g condition. We then verified the microarray data for some of the genes relevant in bone biology using real-time PCR assays and immunoblotting. We confirmed that microgravity downregulated levels of alkaline phosphatase, runt-related transcription factor 2, osteomodulin, and parathyroid hormone receptor 1 mRNA; upregulated cathepsin K mRNA; and did not significantly affect bone morphogenic protein 4 and cystatin C protein levels. The identification of gravisensitive genes provides useful insight that may lead to further hypotheses regarding their roles in not only microgravity-induced bone loss but also the general patient population with similar pathological conditions, such as osteoporosis.

scholarly journals miR-539 mediates osteoblast mineralization by regulating Distal-less genes 2 in MC3T3-E1 cell line

2017 ◽  
Vol 12 (1) ◽  
pp. 294-299 ◽  
Author(s):  
Jianguo Han ◽  
Li Su ◽  
Chunyang Zhang ◽  
Rongcai Jiang
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Cell Line ◽  
Osteoblast Differentiation ◽  
Marker Gene ◽  
Inhibitory Effect ◽  
Negative Regulator ◽  
Expression Level ◽  
Matrix Mineralization ◽  
Marker Gene Expression

AbstractmicroRNAs (miRNAs) play an important role in osteoblast differentiation. However, the mechanisms of miRNAs regulating osteoblast mineralization still needs to be further cleared. Distal-less genes 2 (Dlx2) plays an important role in osteoblast differentiation. We have found that miR-539 was significantly downregulated and Dlx2 was found to be inversely correlated with miR-539 in MC3T3-E1 cell line during osteoblast mineralization. The overexpression of miR-539 significantly decreased the expression level of Dlx2 and suppressed the osteogenic marker gene expression level, alkaline phosphatase activity and matrix mineralization. Our study showed that miR-539 was a negative regulator in osteoblast mineralization and that the targeting of Dlx2 gene partly contributes to this inhibitory effect exerted by miR-539.

Abstract P031: Effect of Exercise on Function and Differentiation of Adipose Tissue Derived Mesenchymal Stromal Cells (MSCs)

Circulation ◽  
2018 ◽  
Vol 137 (suppl_1) ◽  
Author(s):  
Nabanita Kundu ◽  
Cleyton Domingues ◽  
Peter Kokkinos ◽  
Eric Nylen ◽  
Sabyasachi Sen
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Bone Formation ◽  
Aerobic Exercise ◽  
Stromal Cells ◽  
Exercise Intervention ◽  
Diabetic Patients ◽  
Differentiation Potential ◽  
Post Exercise ◽  
Bone Formation Markers

Approximately 422 million people have diabetes worldwide (2014) and it is predicted that diabetes will rise by nearly 54% by 2030. Aerobic exercise is known to show positive effect on health of diabetic and pre diabetic patients. The effect of exercise has been studied extensively using plasma biochemistry but cellular data is scares. Previously, we have shown endothelial progenitor cells (EPCs) can act as a strong cellular biomarker of endothelial function following aerobic exercise as an intervention. In this study, we are examining the effect of aerobic exercise on adipocyte derived MSCs to study stromal cell differentiation and as a cellular surrogate of fat metabolism. Methods: Overweight and obese subjects (n=5) were enrolled in a 12 week exercise intervention study. The biweekly exercise sessions were supervised by a trained exercise physiologist and consisted of a 1 hour sessions that included warm-up and cool-down and 30 min of combined aerobic and resistance training at an exercise intensity of 50-80% of heart rate reserve. Physical and biochemical parameters were tested pre and post exercise. Subcutaneous abdominal fat biopsies were obtained and fat derived stromal cells were cultured in vitro for 2-3 weeks. MSCs were analyzed for mRNA gene expression (qRT-PCR) and cellular oxygen consumption rate (OCR), pre and post 12 week exercise. Results: With exercise, A1C reduced significantly. An increase of METs was also noticed post exercise. Both basal and maximal respiration increased significantly post exercise when compared with commercially obtained MSCs. Simultaneously, mitochondrial genes COX4 and ATP5B (p= 0.01, 1.4 fold, 0.02, 1.5 fold respectively), Glucose transporter, GLUT1 (p=0.04, 1.8 fold), antioxidants GPX3 and CAT (p= 0.01, 3.2 fold and p=0.04, 1.5 fold respectively) upregulated whereas pro-inflammatory cyclo-oxygenase-2 (p=0.04, 2.5 fold) gene reduced significantly, post exercise. Regarding differentiation potential of multipotent MSCs, post exercise, we noted enhanced expression of bone markers such Alkaline Phosphatase (p= 0.03, 2.7 fold) BGLAP and RUNX2 (1.3 and 1.2 fold) and also for collagen marker COL2 (2.4 fold) expression. For adipogenic differentiation potential PPARG mRNA expression was reduced. Interestingly, serum value of osteocalcin increased significantly from 15.0 (5.5) to 16.3(6.1) ng/ml (9% increase, p=0.03) with 1% increase in bone alkaline phosphatase level, post exercise. Conclusion: We conclude that exercise augments cellular glucose transporters (GLUT1), anti-oxidants and reduce MSC inflammation and up-regulates mitochondrial function and gene expression profile of MSCs. Increased serum value of osteocalcin complement increased gene expression of bone formation markers indicating a cross talk between fat derived MSCs and bone formation, post exercise.

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