O8. Use of the novel technique of in situ-reverse transcriptase-polymerase chain reaction to demonstrate vitamin D receptor transcripts in human osteoclasts

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.

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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

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0310123 ◽

2003 ◽

Vol 31 (1) ◽

pp. 123-132 ◽

Cited By ~ 50

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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