Influence of mechanical and biological signals on gene expression in human MG-63 cells: evidence for a complex interplay between hydrostatic compression and vitamin D3 or TGF-β1 on MMP-1 and MMP-3 mRNA levels

2005 ◽  
Vol 83 (1) ◽  
pp. 96-107 ◽  
Author(s):  
V Tasevski ◽  
J M Sorbetti ◽  
S S Chiu ◽  
N G Shrive ◽  
D A Hart
Keyword(s):  
Gene Expression ◽  
Vitamin D ◽  
Polymerase Chain Reaction ◽  
Bone Cells ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tgf Β1 ◽  
In Cells

Biological mediators can influence the activity and differentiation of bone cells. 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3) is known to induce differentiation of precursors into mature osteoblasts, and transforming growth factorβ1 (TGF-β1) can modulate the activity of bone cells leading to alterations in proliferation and gene expression patterns. Bone-derived cells were loaded via intermittent cyclic hydrostatic pressure (icHP) on cells under basal conditions and in the presence of 1,25-(OH)2D3 or TGF-β1. Evaluating the effects of loading on the cells allowed for a comparison to be made between responsiveness to biomechanical and biochemical stimuli and their potential interplay. The effects of icHP on mRNA levels for the specific genes involved in bone remodelling and differentiation were measured in MG-63 cells using reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels for matrix metalloproteinase-1 and -3 (MMP-1 and MMP-3) were significantly, and uniquely, increased (p < 0.001) in cells exposed to icHP under serum-free conditions for 4–12 h. However, mRNA levels for MMP-3, but not MMP-1, were significantly enhanced in cells subjected to static hydrostatic pressure (HP). Treatment of cells with 1,25-(OH)2D3 resulted in increased (p < 0.001) mRNA levels for osteocalcin and decreased (p < 0.001) mRNA levels for both MMP-1 and MMP-3. In cells exposed to icHP and 1,25-(OH)2D3, the mRNA levels for both MMP-1 and MMP-3 were elevated (p < 0.001) compared with hormone alone, but not to the same degree (p < 0.01) as cells subjected to icHP alone. Addition of TGF-β1 to cells led to increases in cell proliferation and expression of collagen I, as well as decreases in expression of osteocalcin and MMP-1 and MMP-3. Exposure of cells to icHP and TGF-β1 again led to unique and significant increases in expression of MMP-1 and MMP-3. No changes in mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or any of the other 9 genes assessed, including those for MMP-2 and MMP-13, were detected under any of the conditions described. Therefore, icHP can induce alterations in mRNA levels for a specific subset of genes in both premature and mature osteoblasts. Such stimuli can modulate the impact of potent biological mediators in defining patterns of gene expression by bone cells and potentially modify function in vivo.Key words: osteoblast, biomechanical loading,1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3), mRNA levels, reverse trans cription-polymerase chain reaction (RT-PCR), transforming growth factor-β1 (TGF-β1).

scholarly journals A beginner’s guide to RT-PCR, qPCR and RT-qPCR

2020 ◽  
Vol 42 (3) ◽  
pp. 48-53 ◽  
Author(s):  
Grace Adams
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Messenger Rna ◽  
Life Science ◽  
Science Research ◽  
Quantitative Real Time Pcr ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Mrna Quantitation ◽  
Polymerase Chain

The development of the polymerase chain reaction (PCR), for which Kary Mullis received the 1992 Novel Prize in Chemistry, revolutionized molecular biology. At around the time that prize was awarded, research was being carried out by Russel Higuchi which led to the discovery that PCR can be monitored using fluorescent probes, facilitating quantitative real-time PCR (qPCR). In addition, the earlier discovery of reverse transcriptase (in 1970) laid the groundwork for the development of RT-PCR (used in molecular cloning). The latter can be coupled to qPCR, termed RT-qPCR, allowing analysis of gene expression through messenger RNA (mRNA) quantitation. These techniques and their applications have transformed life science research and clinical diagnosis.

scholarly journals Ontogeny of erythropoietin gene expression in the sheep fetus: effect of dexamethasone at 60 days of gestation

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 460-466
Author(s):  
GB Lim ◽  
K Jeyaseelan ◽  
EM Wintour
Keyword(s):  
Gene Expression ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Fetal Plasma ◽  
Liver And Kidney ◽  
Polymerase Chain ◽  
High Level ◽  
A Site ◽  
Sheep Fetus

We have used competitive reverse transcription and polymerase chain reaction (RT/PCR) to compare the levels of erythropoietin (Epo) mRNA in the liver and kidneys of the sheep fetus at 60, 80, 100, 130, and 140 days of gestation (term = 145 to 150 days). The effect of dexamethasone infusion in the ewe on Epo gene expression in the 60-day fetus was also investigated. Epo mRNA levels were highest at 60 days of gestation, the earliest age studied, in both liver and kidney. In the liver, Epo mRNA expression declined as gestation proceeded. Kidney Epo mRNA was maintained at a high level until 100 days of gestation, declining significantly in the 130-day fetus (P < .01). Treatment of ewes carrying 60-day fetuses with 0.76 mg/h dexamethasone for 48 hours resulted in a significant decrease in fetal plasma Epo values and Epo mRNA levels in both the liver and kidney. In the dexamethasone-treated fetuses, Epo mRNA in the liver was 52% of control values (P < .05), and in the kidney, 33% of control (P < .001). The results suggest that the kidney may play a more important role as a site of Epo synthesis in the early gestation sheep fetus than previously thought. Glucocorticoids may have a role in the regulation of Epo gene expression.

Novel and sensitive detection systems for the vitamin D receptor — in situ-reverse transcriptase-polymerase chain reaction and immunogold cytochemistry

1996 ◽  
Vol 16 (2) ◽  
pp. 183-195 ◽  
Author(s):  
A P Mee ◽  
L K Davenport ◽  
J A Hoyland ◽  
M Davies ◽  
E B Mawer
Keyword(s):  
Vitamin D ◽  
Polymerase Chain Reaction ◽  
Cell Line ◽  
Vitamin D Receptor ◽  
Human Kidney ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Low Levels

ABSTRACT The receptor for the active metabolite of vitamin D, 1,25(OH)2D3, known as the vitamin D receptor (VDR), belongs to the steroid hormone nuclear receptor superfamily. We have developed novel methods for detection of VDR mRNA and protein within a human promyelomonocytic cell line, HL-60. Using the newly developed technique of in situ-reverse transcriptase-polymerase chain reaction (IS-RT-PCR), low levels of VDR mRNA could be amplified and demonstrated unequivocally within these cells, and also within a human kidney proximal tubule cell line, CL-8. Use of a novel immunogold cytochemical technique has allowed clear and sensitive detection of VDR protein expression within the HL-60 cells. Further development of IS-RT-PCR has allowed us to apply this technique to tissue sections. We have shown clear amplification of VDR transcripts within sections of formalin-fixed paraffin-embedded human kidney and liver. These techniques will be useful to localise specifically the VDR within cell types that contain low levels of mRNA and protein, and will permit further investigation of the role played by 1,25(OH)2D3 in cellular regulatory mechanisms.

scholarly journals Quantification of mRNA for the vitamin D metabolizing enzymes CYP27B1 and CYP24 and vitamin D receptor in kidney using real-time reverse transcriptase- polymerase chain reaction

2003 ◽  
Vol 31 (1) ◽  
pp. 123-132 ◽  
Author(s):  
PH Anderson ◽  
PD O'Loughlin ◽  
BK May ◽  
HA Morris
Keyword(s):  
Vitamin D ◽  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Vitamin D Receptor ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Polymerase Chain

Critical to an understanding of the control of 1,25-dihydroxyvitamin D (1,25D) activity is a molecular appreciation of the regulation of three genes, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), 25-hydroxyvitamin D-24-hydroxylase (CYP24) and vitamin D receptor (VDR). We now report the sensitivity, reproducibility and accuracy of a real-time reverse transcriptase-polymerase chain reaction protocol (Taqman) for the quantification of mRNA levels for these genes in total RNA extracted from kidney tIssue. The sensitivity of the protocol was at least 150 copies of mRNA per reaction. Reproducibility, expressed as the coefficient of variation, ranged between 14 and 30% at the level of approximately 10(4) copies of mRNA per reaction. Accuracy was estimated at greater than 95% for each of these mRNAs. This protocol allows for the comparison of absolute mRNA levels in extracted total RNA in kidneys from animals fed diets containing different levels of calcium, ranging from 0.05% to 1%. Serum 1,25D levels were decreased when the dietary calcium concentration was increased (P<0.05). The levels of CYP27B1 mRNA were highest in the animals fed the 0.05% calcium diet (P<0.01). Conversely, CYP24 and VDR mRNA levels were highest in the animals fed the 1% calcium diet (P<0.01). Both CYP27B1 and CYP24 mRNA levels were major determinants of serum 1,25D levels when dietary calcium intakes were varied in these adult animals (Multiple R(2)=0.70, P<0.01). No significant relationship was detected between kidney CYP27B1 and serum parathyroid hormone (PTH) suggesting that serum calcium may regulate CYP27B1 mRNA expression directly during normocalcaemia. Low levels of CYP24 mRNA were associated with high PTH levels. These findings suggest that kidney CYP24 activity, possibly regulated by factors such as PTH, acts in concert with kidney CYP27B1 to control serum 1,25D levels.

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