|Reinhardt, Timothy - Tim|
Submitted to: Bone
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/8/2000
Publication Date: N/A
Citation: N/A Interpretive Summary: Milk fever is a disease that affects 8-10% of the dairy cows in the U.S., which means that approximately 800,000 cows are affected each year. The combined direct and indirect costs of a milk episode are estimated to exceed $300 per cow. Our research groups' mission is to perform basic and applied research aimed at understanding the underlying causes for the disease and applying these findings to reducing these costs. It is known that vitamin D, working through its receptor (VDR) in bone, intestine and immune cells, is critical to the cow's ability to maintain calcium homeostasis and immune function. This paper describes the development of an antibody for measuring VDR and its applicatio in measuring VDR in bone and bone marrow cells. Of particular importance is the finding of VDR in bone cells that pull calcium out of bone. These cells are specialized cells (osteoclasts) that originate from immune cells (monocytes). This is the clearest evidence presented that these bone osteoclasts are regulated by vitamin D. This is critical to the milk fever cow, as she needs the calcium, from bone, provided by these cells to fight milk fever development and for rapid recovery. The results of these studies will greatly benefit the dairy farmer and dairy industry.
Technical Abstract: 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.