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Vitamin D receptor expression in human lymphocytes. Signal requirements and characterization by western blots and DNA
X P Yu1, H Mocharla, F G Hustmyer
1Section of Endocrinology and Metabolism, Veterans Administration Medical Center, Indianapolis, Indiana 46202.
Abstract:
The signals controlling the expression of the receptor protein for 1 alpha,25-dihydroxyvitamin D3 in normal human lymphocytes and the relationship of this protein to the classical vitamin D receptor were examined. Lymphocytes activated with the OKT3 antibody to the T-cell antigen receptor expressed fewer binding sites as compared to lymphocytes that were activated by the polyclonal activator phytohemagglutinin (PHA). However, combination of OKT3 and phorbol myristate acetate produced a concentration of binding sites similar to the PHA-activated cells. The receptor from OKT3 and OKT3 + phorbol myristate acetate-activated lymphocytes exhibited decreased binding to DNA-cellulose compared to PHA-activated lymphocytes. In lymphocytes activated either by PHA or OKT3 (but not in resting cells), a 50-kDa species cross-reacting with a monoclonal antibody against the intestinal vitamin D receptor was detected. Finally, RNA from activated lymphocytes was amplified by polymerase chain reaction using oligonucleotide primers flanking the 196 base pair long region encoding the DNA-binding domain of the human intestinal receptor. The amplified product showed an identical nucleotide sequence to the DNA-binding domain of the human intestinal receptor. These findings suggest that expression of the 1,25-(OH)2D3 receptor in lymphocytes is triggered by distinct and contingent signals, and that the protein and the mRNA encoding it are identical to the classical vitamin D receptor.
Insights
Signals controlling the 1,25-dihydroxyvitamin D3 receptor in human lymphocytes were studied. Findings indicate distinct signals trigger this receptor, which is identical to the classical vitamin D receptor.
Area of Science:
- Immunology
- Molecular Biology
- Endocrinology
Background:
- The vitamin D receptor (VDR) plays a crucial role in various cellular processes.
- Understanding the regulation of VDR expression in immune cells is important for immune system function.
Purpose of the Study:
- To investigate the signals that control the expression of the 1,25-dihydroxyvitamin D3 receptor (VDR) in human lymphocytes.
- To determine if this lymphocyte VDR is identical to the classical VDR found in other tissues.
Main Methods:
- Lymphocyte activation using OKT3 antibody, phytohemagglutinin (PHA), and phorbol myristate acetate.
- Measurement of VDR binding sites and DNA-cellulose binding.
- Detection of VDR protein using Western blotting with a VDR monoclonal antibody.
- RNA amplification via polymerase chain reaction (PCR) to analyze the VDR DNA-binding domain sequence.
Main Results:
- Lymphocyte activation by PHA resulted in more VDR binding sites compared to OKT3 activation.
- Combined OKT3 and phorbol myristate acetate stimulation yielded VDR binding site levels similar to PHA activation.
- VDR from activated lymphocytes showed reduced DNA-cellulose binding compared to PHA-activated cells.
- A 50-kDa protein cross-reactive with VDR antibody was detected in activated lymphocytes.
- PCR analysis confirmed the mRNA for the VDR DNA-binding domain in activated lymphocytes is identical to the classical VDR.
Conclusions:
- Lymphocyte VDR expression is regulated by distinct and contingent activation signals.
- The VDR protein and its encoding mRNA in lymphocytes are identical to the classical vitamin D receptor.