Characterization of vitamin D receptor immunoreactivity in human bone cells.
The present study examined the expression of the vitamin D receptor (VDR) in adult human bone by immunohistochemical analysis. Antiserum from goats immunized with an N-terminal rat VDR peptide was purified by affinity chromatography. The purified antiserum recognized both endogenous rat and recombinant human VDR in Western blots. The purified antiserum was also able to specifically supershift the recombinant human VDR when analyzed in mobility shift assays. Immunohistochemical analysis of MG-63 cells, a human osteoblastic cell line known to express the VDR, revealed prominent staining over the nuclei of these cells. Immunostaining was greatly attenuated in the presence of an excess of the immunizing peptide. Analysis of bone biopsy samples from 16 normal human subjects immunostained for VDR protein showed strong, immunopositive staining over bone cells, particularly osteoblasts, in keeping with prior studies. In addition, there was significant immunoreactivity observed in nuclei of osteoclasts, lining cells and scattered bone marrow stromal cells of the adult human bone. Results showed that 298 osteoblasts out of 808 (36.9%) examined were immunopositive. It was also observed that 29 osteoclasts out of 125 (23%) contained VDR immunoreactivity. The ability to detect VDR in osteoclasts and stromal cell populations suggests that in addition to regulating osteoblast function, these other cell types are also direct targets of the hormone's action. These results demonstrate the utility of this purified antiserum in detecting the VDR in a variety of molecular techniques and should prove useful in examining receptor expression in various pathological conditions.
Langub
M C
MC
Division of Nephrology, Bone & Mineral Metabolism, University of Kentucky Medical Center, Lexington, KY 40536-0084, USA. clangub@pop.uky.edu
Reinhardt
T A
TA
Horst
R L
RL
Malluche
H H
HH
Koszewski
N J
NJ
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D006041:Goats; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D051381:Rats; D018167:Receptors, Calcitriol / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D017931:DNA Primers; D010446:Peptide Fragments; D018167:Receptors, Calcitriol