|Reinhardt, Timothy - Tim|
Submitted to: Journal of Steroid Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/4/1996
Publication Date: N/A
Citation: Interpretive Summary: Milk fever is a disease which affects 8-10% of the dairy cows in the US, which mean that approximately 800,000 cows are affected each year. The combined direct and indirect costs of a milk fever episode are estimated to exceed $300 per cow. Our research group's mission is to perform basic and applied research aimed at reducing productivity losses and complications that necessitate further treatment of these animals with antibiotics and drugs which we would like to avoid. The vitamin D receptor plays a pivotal role in milk fever prevention. Therefore, a basic understanding of its function is necessary to understand milk fever. The present study demonstrates that the vitamin D receptor can regulate expression of important functions in milk fever by two mechanisms. The first involves vitamin D, its receptor, vitamin A receptor, and vitamin A acid. The second needs only vitamin D and its receptor. Understanding the significance of these two mechanisms should help reduce the incidence of milk fever; thus, saving dairy producers millions of dollars. The consumers are the eventual beneficiaries of this work, as they are further assured of economical and wholesome products.
Technical Abstract: The nature of the DNA-binding interactions of the human vitamin D receptor (hVDR) with the murine osteopontin vitamin D response element (mOP VDRE) was examined. Both recombinant hVDR and human retinoid X receptor beta (hRXRB) proteins were obtained from baculovirus-infected Sf9 insect cells. Mixing extracts of the two recombinant proteins resulted in the strong, specific formation of a slower migrating complex in the electrophoretic mobility shift assay. Crude extracts of the expressed hVDR alone were also capable of binding with high affinity to the mOP sequence, and this binding was enhanced in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Competition experiments confirmed the specificity of this interaction and revealed that the human osteocalcin VDRE was a poor competitor for this binding. Ethylation interference footprint analyses of hVDR/hRXRB and hVDR complexes revealed only subtle differences in how these two different VDR-containing complexes interacted with the mOP VDRE. The footprints displayed contact points in both halves of the direct repeat format, confirming the dimeric and major groove interactions of both types of complexes. DNA affinity chromatography of labeled hVDR extracts revealed a peak eluting at ca. 290 mM KCl that was capable of rebinding to the mOP sequence in gel shift experiments.