METABOLISM AND FUNCTION OF FAT SOLUBLE VITAMINS

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0027332
• Project Start Date: Jan 1, 1960
• Project End Date: Sep 30, 2013
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
BIOCHEMISTRY

Non Technical Summary
(N/A)

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	3999	1000	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
3999 - Animal research, general;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

nutrient function

animal nutrition

metabolites

enzymes

vitamins

biological activity

vitamin d

vitamin a

metabolism

mechanism of action

identification

synthesis

hormones

biochemistry

nutrient deficiency

molecular biology

Goals / Objectives
Delineate at the molecular level the metabolism and mechanism of action of vitamins A and D, and to determine hormones and nutritional factors which modulate their activity.

Project Methods
The primary approach is to synthesize radioactive vitamin A and vitamin D compounds, inject them into vitamin deficient animals, extract their tissues anddetermine by chromatographic methods the compounds to which they are converted. Once new metabolities are identified, they are isolated in pure form and their structures determined by mass spectrometry and nuclear magnetic resonance spectrometry. The structures of these compounds are then prepared by chemical synthesis and their biological activity determined in animals. Their use in agriculture and medicine are also tested when appropriate. The tissue conversions bringing about these compounds are searched for at the enzymatic or subcellular level; the enzymes are isolated and studied from a physical-chemicalpoint of view. The mechanism of action of the final target hormone in the target tissue is determined by molecular biological techniques.

Progress 01/01/60 to 09/30/13

Outputs
OUTPUTS: It is well established that the incidence of multiple sclerosis is very low at the equator without attendant hypercalcemia. This triggered an investigation of the effect of ultraviolet light on the incidence of the experimental model of multiple sclerosis (MS). The results show that ultraviolet light suppresses the experimental form of MS independent of vitamin D production. This focuses on the potential of ultraviolet light itself as a therapeutic regimen for reducing the incidence of MS. Several new analogs of 1 alpha,25-dihydroxyvitamin D3(1,25-(OH)2D3) have been chemically synthesized. With them, we can demonstrate the lack of need for a CD-ring for vitamin D activity and furthermore we have prepared analogs that demonstrate that the 1 alpha-hydroxylase is unable to act on 2-methylene-19-nor-(20S)-dihydroxyvitamin D3 to produce the 1 alpha-hydroxylated derivative. Thus, the 1-deoxy compound will not be of any therapeutic value since it cannot be 1-hydroxylated. The use of 1 alpha-hydroxylated vitamin D compounds to treat experimental acne has revealed that a subset of side chain-deleted forms of 1,25-(OH)2D3 are effective in correcting acne, whereas 1,25-(OH)2D3 analogs with a full side chain cannot. This opens up potential therapy of acne with vitamin D compounds. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The most important impact of current work is the ultraviolet light can be used experimentally to reduce the incidence of multiple sclerosis in an animal model. A clinical trial will therefore have to be performed to verify this finding in humans. We now have analogs of 1,25-(OH)2D3 that can treat the experimental model of acne. This opens up the possibility of a vitamin D compound for be used in the treatment of this disorder.

Publications

• Plum, L.A. and DeLuca H.F. 2010. Vitamin D, disease and therapeutic opportunities. Nature Rev./Drug Discovery 9:941-955.
• Zhu, J., Barycki, R., Chiellini G. and DeLuca H.F. 2010 Screening of selective inhibitors of 1 alpha,25-dihydroxyvitamin D3 24-hydroxylase using recombinant human enzyme expressed in Escherichia coli. Biochemistry 49:10403-10411.
• Plum, L.A. and DeLuca, H.F. 2010 The functional metabolism and molecular biology of vitamin D action. In: Vitamin D: Physiology, Molecular Biology, and Clinical Applications, second edition (Holick, M.F., ed.), Human Press/Springer Science Business Media, LLC, New York, pp. 61-97.
• Sokolowska, K., Mourino, A., Sicinski, R.R., Sigueiro, R., Plum, L.A. and DeLuca H.F. 2010. Synthesis and biological evaluation of 6-methyl analog of 1 alpha,25-dihydroxyvitamin D3. J. Steroid Biochem. Mol. Biol. 121:29-33.
• Glebocka, A., Sicinski, R.R., Plum, L.A. and DeLuca H.F. 2010. New 1 alpha,25-dihydroxy-19-norvitamin D3 analogs with a frozen A-ring conformation. J. Steroid Biochem. Mol. Biol. 121:46-50.
• Sibilska, I., Barycka, K.M., Sicinski, R.R., Plum, L.A. and DeLuca H. F. 2010. 1-Desoxy analog of 2MD: Synthesis and biological activity of (20S)-25-hydroxy-2-methylene-19-norvitamin D3. J. Steroid Biochem. Mol. Biol. 121:51-55.
• Schubert, L. and DeLuca H.F. DeLuca. 2010. Hypophosphatemia is responsible for skeletal muscle weakness of vitamin D deficiency. Arch. Biochem. Biophys. 500:157-161.
• Wang, Y., Becklund, B. R. and DeLuca, H. F. 2010. Identification of a highly specific and versatile vitamin D receptor antibody. Arch. Biochem. Biophys. 494:166-177.
• Becklund, B.R., Severson, K.S., Vang, S.V. and DeLuca, H.F. 2010. UV radiation suppresses experimental autoimmune encephalomyelitis independent of vitamin D production. Proc. Natl. Acad. Sci. USA 107:6418-6423.
• Nieves, N.J., Ahrens, J. M., Plum, L.A., DeLuca, H.F. and Clagett-Dame, M. 2010. Identification of a unique subset of 2-methylene-19-nor analogs of vitamin D with comedolytic activity in the rhino mouse. J. Invest. Dermatol. 130:2359-2367.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Two important advances on the structure and function activities of vitamin D compounds has been completed. We have designed, synthesized, and tested a form of 1alpha,25-dihydroxyvitamin D3 that has the C,D-ring replaced with a hydrocarbon linkage. This compound is able to bind the vitamin D receptor, although one order of magnitude less effectively than the natural hormone and can elicit biological responses both in vitro and in vivo in raising serum calcium. These results clearly demonstrate that the C,D-ring is not essential for the functions of the active form of vitamin D. We have removed the C-20-methyl group from 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3, the most potent analog we have yet prepared. Removal specifically of the C-20-methyl selectively eliminates the ability of the vitamin D compounds to mobilize calcium from bone. Thus, this compound retains full activity in binding to the receptor, in cellular differentiation in HL-60 cells, and in stimulating intestinal calcium transport. However, it has virtually no activity in mobilizing calcium from bone. These results demonstrate the selectivity of this analog and may be useful in circumstances where raising blood calcium at the expense of bone is not desirable, as for example in osteoporosis. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The outcome of the first synthesis is to demonstrate that we can greatly modify the vitamin D structure and still retain biological activities. The second synthesis demonstrates that we can selectively eliminate specific actions of vitamin D by selective modification of the structure and still retain its full biological activity in other responses. This adds additional credence to the belief that we can synthesize compounds that will selectively carry out single functions of the vitamin D molecule without affecting unwanted functions such as the rise in serum calcium. These analogs, especially the second analog, will positively be very useful in the treatment of bone loss diseases such as osteoporosis.

Publications

• Barycki, R., Sicinski, R.R., Plum, L.A., Grzywacz, P., Clagett-Dame, M. and DeLuca, H.F. 2009. Removal of the 20-methyl group from 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 (2MD) selectively eliminates bone calcium mobilization activity. Bioorg. & Med. Chem. 17:7658-7669.
• Plonska-Ocypa, K., Sicinski, R.R., Plum, L.A., Grzywacz, P., Frelek, J., Clagett-Dame, M. and DeLuca, H.F. 2009. 3-Methyl-substituted des-C,D analogs of (20S)-1alpha,25-dihydroxy-2-methylene-19-norvitamin D3 (2MD): synthesis and biological evaluation. Bioorg. Med. Chem. 17:1747-1763.
• Becklund, B.R., James, B.J., Gagel, R.F. and DeLuca, H.F. 2009. The calcitonin/calcitonin gene related peptide-alpha gene is not required for 1alpha,25-dihydroxyvitamin D3-mediated suppression of experimental autoimmune encephalomyelitis. Arch. Biochem. Biophys. 488:105-108.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: We have continued to pursue the molecular mechanism whereby 1,25-dihdyroxyvitamin D3 (1,25-(OH)2D3) increases intestinal calcium absorption. A calcium channel protein, TRPV6, is strongly induced by 1,25-(OH)2D3. However, mice that have a null mutation in this gene respond equally well as Wild-type mice to 1,25-(OH)2D3 in the elevation of calcium absorption and in the rise in serum calcium. This clearly demonstrates that neither TRPV6 nor calbindin D9k (shown previously) are required for the vitamin D activation of calcium absorption. We previously found that calcium in the diet is required for 1,25-(OH)2D3 to suppress experimental autoimmune encephalomyelitis (EAE) the animal model of multiple sclerosis. Further, hypercalcemia is associated with 1,25-(OH)2D3 suppression of EAE. Calcitonin (CT) is secreted in response to hypercalcemia. CT administration reduces the requirement of 1,25-(OH)2D3 to suppress EAE. A combination of CT and low doses of 1,25-(OH)2D3 can suppress EAE without hypercalcemia, suggesting that a combination of these hormones may be an excellent therapy to suppress multiple sclerosis. PARTICIPANTS: For the first half of this report, no graduate student was involved and the experimental work was directed by Professor DeLuca. However, the demonstration that calcitonin together with low doses of vitamin D can suppress experimental autoimmune encephalomyelitis or a model of multiple sclerosis involved an excellent graduate student, Bryan Becklund. He contributed to this work by very conscientious experimental work and follow-up. Furthermore, I believe this project was the vehicle for training of Mr. Becklund whom I believe will be a contributing scientist in the future. Finally, the demonstration that calcitonin together with low dose of 1,25-(OH)2D3 or an analog to suppress multiple sclerosis could result in a practical pharmaceutical approach for the treatment of this disease. Additional developmental work will be required before we can make such a statement however. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
These studies more clearly define the role and sites of action of the hormonal form of vitamin D and its analogs. These will be used in the development of specific vitamin D therapies for certain disorders such as MS.

Publications

• Nakabayashi, M., Yamada, S., Yoshimoto, N., Tanaka, T., Igarashi, M., Ikura, T., Ito, N., Makishima, M., Tokiwa, H., DeLuca, H.F., and Shimizu, M. 2008. Crystal structures of rat vitamin D receptor bound to adamantyl vitamin D analogs: structural basis for vitamin D receptor antagonism and partial agonism. J. Med. Chem. 51(17):5320-5329.
• DeLuca, H.F. 2008. Evolution of our understanding of vitamin D. Nutrition Rev. 66(Suppl. 2):S73-S87.
• Williams, K.B., and DeLuca, H.F. 2008. 2-Methylene-19-nor-20(S)-1alpha-hydroxy-bishomopregnacalciferol [20(S)-2MbisP], an analog of vitamin D3 [1,25(OH)2D3], does not stimulate intestinal phosphate absorption at levels previously shown to suppress parathyroid hormone. Steroids 73:1277-1284.
• Kutuzova, G.D., Sundersingh, F., Vaughan, J., Tadi, B.P., Ansay, S.E., Christakos, S., and DeLuca H.F. 2008. TRPV6 is not required for 1alpha,25-dihydroxyvitamin D3-induced intestinal calcium absorption in vivo. Proc. Natl. Acad. Sci. USA 105:19655-19659.
• Shimizu, M., Miyamoto, Y., Takaku, H., Matsuo, M., Nakabayashi, M., Masuno, H., Udagawa, N., DeLuca, H.F., Ikura, T., and Ito, N. 2008. 2-Substituted-16-ene-22-thia-1alpha,25-dihydroxy-26,27-dimethyl-19-no rvitamin D3 analogs: synthesis, biological evaluation, and crystal structure. Bioorg. Med. Chem. 16:6949-6964.
• Liu, N., Nguyen, L. Chun, R.F., Lagishetty, V., Ren, S., Wu, S., Hollis, B., DeLuca, H.F., Adams, J.S., and Hewison, M. 2008. Altered endocrine and autocrine metabolism of vitamin D in a mouse model of gastrointestinal inflammation. Endocrinology 149(10):4799-4808.
• Takaku, H, Miyamoto, Y., Asami, S., Shimazaki, M., Yamada, S., Yamamoto, K., Udagawa, N., DeLuca, H.F., and Shimizu, M. 2008. Synthesis and structure-activity relationships of 16-ene-22-thia-1alpha,25-dihydroxy-26,27-dimethyl-19-norvitamin D3 analogs having side chains of different sizes. Bioorg. Med. Chem. 16:1796-1815.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: We have developed a null-mutant mouse that does not express calbindin D9k, a protein that reputedly has been the carrier in intestinal calcium absorption stimulated by vitamin D. Our results clearly demonstrate that this protein is not required for the intestine to respond to the active vitamin D hormone in terms of intestinal calcium transport, placing considerable doubt on its participation in this mechanism. We have also developed a mouse in which the CYP27B1 (1α-hydroxylase) has been replaced with a lac-Z gene. These mutant mice do not express the 1α-hydroxylase enzyme and have characteristics exactly as the vitamin D dependency rickets Type II in human beings. Interestingly, under calcium deficiency or vitamin D deficiency conditions, the lac-Z gene is strongly expressed in the proximal convoluted tubule cells of the kidney and in placenta of pregnant mice but is not expressed in skin, casting doubt on the so-called paracrin function of vitamin D in skin. Lithocholic acid has been reported to be a ligand for the vitamin D receptor. Vitamin D-deficient rats were tested as to whether their intestinal calcium transport and serum calcium elevation could be expressed by lithocholic acid dosage. In vitamin D-deficient mice, lithocholic acid markedly increased serum calcium concentrations, very much as a vitamin D-active compound but is unable to increase serum calcium in vitamin D-sufficient animals, suggesting that although lithocholic acid can act as an analog, it is a poor competitor to the native vitamin D hormone and, therefore, the vitamin D system is not a bile acid sensor as has been suggested. PARTICIPANTS: Post Docs: Kasia Barycka, Rafal Barycki, Agnieszka Glebocka, Pawel Gryzwacz, Agnieszka Szczepanska, Padmaja Tadi Graduate Students: Bryan Becklund, Gina Gialamas, James Kim TARGET AUDIENCES: Biochemical, biophysical and chemical scientific research communities with the intent of increasing knowledge.

Impacts
These studies more clearly define the role and sites of action of the hormonal form of vitamin D. This will be in the development of specific vitamin D therapies for skin and intestinal disorders.

Publications

• DeLuca, H.F., Plum, L.A., and Clagett-Dame, M. (2007). Selective Analogs of 1α,25-dihydroxyvitamin D3 for the Study of Specific Functions of Vitamin D. J. Steroid Biochem. Mol. Biol. 103:263-268.
• Sicinski, R.R., Glebocka, A., Plum, L.A. and DeLuca, H.D. (2007). An Analog of 1α-Dihydroxy-19-Norvitamin D3 with 1α-Hydroxy Group Fixed in the Axial Position Lacks Biological Activity in vitro. J. Steroid Biochem. Mol. Biol. 103:293-297.
• Glebocka, A., Sicinski, R.R., Plum, L.A., and DeLuca, H.F. (2007). 2-(3'-Hydroxypropylidene)-1α-Hydroxy-19-Norvitamin D Compounds with Truncated Side Chains. J. Steroid Biochem. Mol. Biol. 103:310-315.
• Nehring, J.A., Zierold, C., and DeLuca, H.F. (2007). Lithocholic acid can carry out in vivo functions of vitamin D. Proc. Natl. Acad. Sci. USA 104:10006-10009.
• Williams, K.B., and DeLuca, H.F. (2007). Characterization of Intestinal Phosphate Absorption Using a Novel in vivo Method. Am. J. Physiol. Endocrinol. Metab. 292:E1917-1921.
• Zierold, C., Nehring, J.A., and DeLuca, H.F. (2007). Nuclear Receptor 4A2 and C/EDPβ Regulate the Parathyroid Hormone-Mediated Transcriptional Regulation of the 25-Hydroxyvitamin D3-1α-Hydroxylase. Arch. Biochem. Biophys. 460:233-239.
• Grzywacz, P., Plum, L.A., Sicinski, R.R., Clagett-Dame, M., and DeLuca, H.F. (2007). Methyl Substitution of the 25-Hydroxy Group on 2-Methylene-19-nor-1α,25-Dihydroxyvitamin D3 (2MD) Reduces Potency but Allows Bone Selectivity. Arch. Biochem. Biophys. 460:274-284.
• Akhter, S., Kutuzova, G.D., Christakos, S., and DeLuca, H.F. (2007). Calbindin D9k is not Required for 1,25-Dihydroxyvitamin D3-Mediated Ca2+ Absorption in Small Intestine. Arch. Biochem. Biophys. 460:227-232.
• Vanhooke, J.L., Padmaja Taxi, B., Benning, M.M., Plum, L.A., and DeLuca, H.F. (2007). New Analogs of 2-Methylene-19-Nor-(20S)-1,25-Dihydroxyvitamin D3 with Conformationally Restricted Side Chains: Evaluation of Biological Activity and Structural Determination of VDR-Bound Conformations. Arch. Biochem. Biophys. 460:161-165.
• Thomson, B., Ahrens, J.M., Ntambi, J.M., DeLuca, H.F., and Clagett-Dame, M. (2007). 2-Methylene-19-Nor-1α-Hydroxyvitamin D3 Analogs Inhibit Adipocyte Differentiation and PPARγ2 Gene Transcription. Arch. Biochem. Biophys. 460, 192-201.

Progress 01/01/06 to 12/31/06

Outputs
A new series of 2-methylene-19-nor-1?,25-dihydroxyvitamin D3 derivatives have been synthesized which lack the 26- or 27-methyl groups in an attempt to reduce calcemic activity and emphasizing its ability to suppress parathyroid hormone synthesis and secretion. A total of 6 analogs were produced; all are superior to the best currently marketed vitamin D compounds for dialysis patients. The patents have been filed on these substances. We have also prepared two new analogs, i.e. 2-(3'-alkoxypropylidene-19-nor-1?,25-dihydroxyvitamin D3 (Z-isomer) and its 20S isomer that are selectively active on intestine. Continued work on 2MD strongly supports its anabolic activity on bone. Currently, 2MD is in Phase II clinical evaluation for the treatment of osteoporosis.

Impacts
These new derivatives of 1,25-dihydroxyvitamin D3 will continue to lead to new therapeutics for treatment of a variety of diseases.

Publications

• Vanhooke, J.L., Prahl, J.M., Kimmel-Jehan, C., Mendelsohn, M., Danielson, E.W., Healy, K.D. and DeLuca, H.F. 2006. CYP27B1 Null Mice with LacZ Reporter Gene Display No 25-Hydroxyvitamin D3-1?-Hydroxylase Promoter Activity in the Skin. Proc. Natl. Acad. Sci. USA 103:75-80.
• Albert, D.M., Plum, L.A., Yang, W., Marcet, M., Lindstrom, M.J., Clagett-Dame, M. and DeLuca, H.F. 2005. Responsiveness of human retinoblastoma and neuroblastoma models to a non-calcemic 19-nor vitamin analog. J. Steroid Biochem. Mol. Biol. 97:165-172.
• Plum, L.A., Fitzpatrick, L.A. Ma, X., Binkley, N.C., Zella, J.B., Clagett-Dame, M. and DeLuca, H.F. 2006. 2MD, a new anabolic agent for osteoporosis treatment. Osteoporosis Int. 17:704-715.

Progress 01/01/05 to 12/31/05

Outputs
During the past year, we have filed approximately 15 patents on new forms of vitamin D that have been synthesized in the laboratory. Perhaps the most important of the analogs made during the past year is the 2-methylene-17,20-dehydro-1alpha,25-dihydroxy-19-nor-vitamin D3 compounds. We have made both the cis- and trans-configurations. The most interesting finding is that the cis-configuration or Z-isomer is extremely active in virtually every aspect of vitamin D function, especially in the newly discovered bone synthesis function. On the other hand, the E-isomer has very little calcemic activity but retains strong transcriptional activity and is very active in cellular differentiation. These analogs demonstrate very clearly the chemical synthetic capability of separating the functions of vitamin D. From this discovery will flow analogs that are specific for bone versus those that are specific for treatment of the immune system or in treatment of cancer. A continued follow-up of experimental autoimmune encephalomyelitis (EAE), a disease resembling multiple sclerosis in man has been the demonstration that the rise in serum calcium which occurs in response to the vitamin D analogs tested to date is at least in part responsible for the curative action in this disease. Thus, in female mice, the hypercalcemia is largely responsible for prevention of EAE. This is not true in the case of the male. A new and important advance has been made with the production of a mutant mouse that lacks the 1alpha-hydroxylase enzyme responsible for converting 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3. Instead, the promoter expresses a gene expressing the beta-galactosidase gene. By following the beta-galactosidase, we can now conclusively demonstrate that under normal conditions, skin keratinocytes or any cell in the skin do not express the 1alpha-hydroxylase even in response to severe hypocalcemia. This counters the claim that the skin possesses its own mechanism for producing the active form of vitamin D.

Impacts
The new analogs of 1,25-dihydroxyvitamin D will lead to specific new therapeutics for treatment of a variety of diseases.

Publications

• DeLuca, H.F., Plum, L.A., Clagett-Dame, M., Shevde, N.K. and Pike, J.W. 2005. 2-Carbon-modified analogs of 19-nor-1alpha,25-dihydroxyvitamin D3. In: Vitamin D, 2nd Edition (Feldman, D., J.W. Pike, and F.H. Glorieux, eds.), Chapter 87, pp.1543-1555 (Elsevier Academic Press, Burlington, MA (2005).
• Healy, K.D., Vanhooke, J.L., Prahl, J.M. and DeLuca, H.F. 2005. Parathyroid hormone decreases renal vitamin D receptor expression in vivo. Proc. Natl. Acad. Sci. USA 102:4724-4728.
• Ke, H.Z., Qi, H., Crawford, D.T., Simmons, H.A., Xu, G., Li, M., Plum, L., Clagett-Dame, M., DeLuca, H.F., Thompson, D.D. and Brown, T.A. 2005. A new vitamin D analog, 2MD, restores trabecular and cortical bone mass and strength in ovariectomized rats with established osteopenia. J. Bone Miner. Res. 20:1742-1755.
• Meehan TF, Vanhooke, J., Prahl, J. and DeLuca, H.F. 2005. Hypercalcemia produced by parathyroid hormone suppresses experimental autoimmune encephalomyelitis in female but not male mice. Arch. Biochem. Biophys. 442:214-221.
• Healy, K.D., Frahm, M.A. and DeLuca, HF. 2005. 1,25-Dihydroxyvitamin D3 up-regulates the renal vitamin D receptor through indirect gene activation and receptor stabilization. Arch. Biochem. Biophys. 433: 466-473.
• DeLuca, H. F. 2005. 2005. Historical perspective. In: Vitamin D, 2nd Edition (Feldman, D., J.W. Pike, and F.H. Glorieux, eds.), Chapter 1, pp. 3-12, Elsevier Academic Press, Burlington, MA.

Progress 01/01/04 to 12/31/04

Outputs
The ligand binding domain (LBD) of the vitamin D receptor (VDR) has been crystallized with several analogs of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) ranging in biological activity from little calcemic activity to compounds that are very strong in the construction of bone. We conclude that these analogs do not differ in function by differing in altering 3-dimensional structure of the crystalline VDR. It is, however, possible that the 3-dimensional structure in solution might be differentially affected by these analogs. The VDR level in the kidney is very markedly regulated by the serum calcium level and with the presence of 1,25-(OH)2D3. Under hypocalcemic conditions, the VDR is not expressed in the kidney and parathyroid. The administration of calcium alone or 1,25-(OH)2D3 alone will not elevate the receptor level. However, when both are given, marked elevations of the VDR are noted. Under conditions of hypocalcemia, the activated VDR is unable to stimulate the vitamin D degradation enzyme, namely the CYP-24 and is unable to shut down the 1a-hydroxylase resulting in high blood levels of 1,25-(OH)2D3 which in turn functions to elevate serum calcium. 2-Alkylidene modified 19-nor-1,25-(OH)2D3 analogs have been discovered that selectively stimulate intestinal calcium absorption without stimulating bone calcium mobilization.

Impacts
These compounds may be useful in the treatment of intestinally-based diseases such as inflammatory bowel disease.

Publications

• Vanhooke, J.L., Benning, M.M., Bauer, C.B., Pike, J.W. and DeLuca, H.F. 2004. Molecular structure of the rat vitamin D receptor ligand binding domain complexed with 2-carbon-substituted vitamin D3 hormone analogues and a LXXLL-containing coactivator peptide. Biochemistry 43:4101-4110 .
• Plum, L.A., Prahl, J.M., Ma, X., Sicinski, R.R., Gowlugari, S., Clagett-Dame, M. and DeLuca H.F. 2004. Biologically active noncalcemic analogs of 1a,25-dihydroxyvitamin D with an abbreviated side chain containing no hydroxyl. Proc. Natl. Acad. Sci. USA 101:6900-6904.
• Binkley, N., Krueger, D., Cowgill, C.S., Plum, L., Lake, E., Hansen, K.E., DeLuca, H.F. and Drezner, M.K. 2004. Assay variation confounds the diagnosis of hypovitaminosis D: A call for standardization. J. Clin. Endocrinol. Metab. 88:3152-3157.
• Grzywacz, P., Plum, L.A., Sicinska, W., Sicinski, R.R., Prahl, J.M. and DeLuca, H.F. 2004. 2-Methylene analogs of 1a-hydroxy-19-norvitamin D3: Synthesis, biological activities and docking to the ligand binding domain of the rat vitamin D receptor. J. Steroid Biochem. Mol. Biol. 89-90:13-17.

Progress 01/01/03 to 12/31/03

Outputs
New Analogs of 1a,25-Dihydroxyvitamin D3 have been prepared that are essentially devoid of calcemic activity while retaining full ability to activate transcription in vitro. These compounds are: 2-methylene-19-nor-1a-hydroxypregnacalciferol, 2-methylene-19-nor-1a-hydroxyhomopregnacalciferol, and 2-methylene-19-nor-20(S)-bishomopregnacalciferol. The most exciting of the three is the 20(S)-bishomo compound that is devoid of calcemic activity when dosed orally to rats as high as 300 micrograms/kg body weight. On the other hand to show that it is active in vivo, total parathyroid hormone suppression takes place at approximately 9 micrograms/kg body weight. This compound, therefore, appears ideal for the treatment of patients suffering from kidney failure and on hemodialysis. A retinoic acid-induced protein has been discovered in the developing embryo and is present at exactly the time when patterning is occurring in the developing hindbrain. A role for retinoic acid in patterning the entire axial skeleton was also discovered. Studies of the retinoid, 4-hydroxyphenyl retinamide, shows it is hydrolyzed to retinoic acid in vivo.

Impacts
This work is important because for the first time, we have discovered a truly non-calcemic vitamin D compound that is fully able to function in vivo to suppress the parathyroid gland and the preproparathyroid hormone synthesis. This compound would be an ideal agent to treat dialysis patients who have developed secondary hyperparathyroidism. In short, we believe this compound to be a third-generation pharmaceutical for this purpose.

Publications

• Zella, J.B. and DeLuca, H.F. 2003. Vitamin D and autoimmune diabetes. J. Cell. Biochem. 88:216-222.
• Zierold, C., Mings, J.A. and DeLuca, H.F. 2003. Regulation of 25-hydroxyvitamin D3-24-hydroxylase mRNA by 1,25-dihydroxyvitamin D3 and parathyroid hormone. J. Cell. Biochem. 88:234-237.
• Rohde, C.M. and DeLuca, H. 2003. Bone resorption activity of all-trans retinoic acid is independent of vitamin D in rats. J. Nutr. 133:777-783.
• Grzywacz, P., Sicinski, R.R. and DeLuca, H.F. 2003. A concise synthesis of an AHR endogenous ligand with the indolecarbonylthiazole skeleton. Hereocycles 60:1219-1224.
• Healy, K.D., Zella, J.B., Prahl, J.M. and DeLuca, H.F. 2003. Regulation of the murine renal vitamin D receptor by 1,25-dihydroxyvitamin D3 and calcium. Proc. Natl. Acad. Sci. USA 100:9733-9737.
• Zella, J.B., McCary, L.C. and DeLuca, H.F. 2003. Oral administration of 1,25-dihydroxyvitamin D3 completely protects NOD mice from insulin-dependent diabetes mellitus. Arch. Biochem. Biophys. 417:77-80.
• Kaiser, M., Merrill, R., Stein, A., Breburda, E. and Clagett-Dame, M. 2003. Vitamin A deficiency in the late gastrula stage rat embryo results in a one to two vertebral anteriorization that extends throughout the axial skeleton. Dev. Biol. 257:14-29.
• Chapman, J. S., Weiss, K.L., Curley, R.W., Highland, M.A., Clagett-Dame, M. 2003. Hydrolysis of 4-HPR to atRA occurs in vivo but is not required for retinamide-induced apoptosis. Arch. Biochem. Biophys. 419:234-243.

Progress 01/01/02 to 12/31/02

Outputs
2-Carbon-modified analogs of 19-nor-1alpha ,25-dihydroxyvitamin D3 have been prepared. One of these (2-methylene-19-nor-20S-1,25-dihydroxyvitamin D3) is a bone specific analog that is being developed for treatment of osteoporosis. Another is 2-methylene-1alpha-hydroxy-bishomo-(20S)-19-nor-vitamin D3 (2BMP) that has no calcium activity but causes differentiation of skin cells and suppression of parathyroid glands. It is being developed for treatment of renal osteodystrophy and psoriasis.

Impacts
The metabolically active form of vitamin D and its precursor were isolated, identified and synthesized along with many analogs resulting in 7 major therapies for bone disease world-wide. The vitamin D endocrine system was delineated and the mechanism of action of 1,25-dihydroxyvitamin D3 was elucidated proving valuable information for therapy. The role of vitamin K in causing gamma carboxylation of glutamate residues of clotting proteins and how the anticoagulant coumadin functions was discovered allowing a more thorough understanding of blood-clotting mechanisms.

Publications

• Sicinski, R. R., P. Rotkiewicz, A. Kolinski, W. Sicinska, J. M. Prahl, C. M. Smith, and H. F. DeLuca. (2002) 2-Ethyl and 2-Ethylidene Analogues of 1alpha,25-Dihydroxy-19-Norvitamin D3: Synthesis, Conformational Analysis, Biological Activities, and Docking to the Modeled rVDR Ligand Binding Domain. J. Med. Chem. 45, 3366-3380.
• Zierold, C., J. A. Mings, J. M. Prahl, G. G. Reinholz, and H. F. DeLuca. (2002) Protein Synthesis is Required for Optimal Induction of 25-Hydroxyvitamin D3-24-Hydroxylase, Osteocalcin, and Osteopontin mRNA by 1,25-Dihydroxyvitamin D3. Arch. Biochem. Biophys. 404, 18-24.
• Qi, X., R. Pramanik, J. Wang, R. M. Schultz, R. K. Maitra, J. Han, H. F. DeLuca, and G. Chen. (2002) The p38 and JNK Pathways Cooperate to trans-Activate Vitamin D Receptor via c-Jun/AP-1 and Sensitize Human Breast Cancer Cells to Vitamin D3-induced Growth Inhibition. J. Biol. Chem. 277, 25884-25892.
• Clagett-Dame, M. and H.F. DeLuca. (2002) The Role of Vitamin A in Mammalian Reproduction and Embryonic Development. Annu. Rev. Nutr. 22, 347-381.
• Shevde, N. K., L. A. Plum, M. Clagett-Dame, H. Yamamoto, J. W. Pike, and H. F. DeLuca. (2002) A Potent Analog of 1alpha,25-Dihydroxyvitamin D3 Selectively Induces Bone Formation. Proc. Natl. Acad. Sci. USA 99, 13487-13491.
• Song, J., M. Clagett-Dame, R.E. Peterson, M.E. Hahn, W.M. Westler, R.R. Sicinski, and H.F. DeLuca. (2002) A Ligand for the Aryl Hydrocarbon Receptor Isolated from Lung. Proc. Natl. Acad. Sci. USA 99,14694-14699.
• Meehan, T. F. and H. F. DeLuca. (2002) The Vitamin D Receptor is Necessary for 1alpha,25-Dihydroxyvitamin D3 to Suppress Experimental Autoimmune Encephalomyelitis in Mice. Arch. Biochem. Biophys. 408, 200-204.
• Sicinski, R. R., J. M. Prahl, C. M. Smith, and H. F. DeLuca. (2002) New Highly Calcemic 1alpha, 25-Dihydroxy-19-Norvitamin D3 Compounds with Modified Side Chain: 26,27-Dihomo- and 26,27-Dimethylene Analogs in 20S-Series. Steroids 67, 247-256.
• Meehan T. F. and H. F. DeLuca. (2002) CD8+ T Cells are not Necessary for 1alpha,25-Dihydroxyvitamin D3 to Suppress Experimental Autoimmune Encephalomyelitis in Mice. Proc. Natl. Acad. Sci. USA 99, 5557-5560.

Progress 01/01/01 to 12/31/01

Outputs
This group discovered that vitamin D must be metabolically activated before it could function and then proceeded to isolate and identify 25-hydroxyvitamin D3 as the main circulating form of vitamin D and the 1,25-dihydroxyvitamin D3 as the vitamin D active form. The group continued to isolate and identify metabolites registering some 30 metabolites of vitamin D. Furthermore, they have carried out the synthesis of well over 300 analogs of 1,25-dihydroxyvitamin D3. This activity has resulted in the marketing of at least 7 major drugs used world-wide for the treatment of renal osteodystrophy and osteoporosis along with some other minor indications providing earnings for UW-Madison of approximately $130 million to date. A nuclear vitamin D receptor (VDR) was discovered by this and another group. This receptor was cloned and its structure and the gene structure determined. The Wisconsin group elucidated the existence of the vitamin D endocrine system based in the kidney and has elucidated its major regulators and their mechanisms of action. The Wisconsin group in competition with other groups has successfully elucidated the vitamin D responsive elements found in a number of responsive genes and has provided the outline of its molecular mechanism of action in carrying out these functions. The finding of VDR in tissues not associated with the maintenance of calcium and phosphorus and bone led to the discovery that vitamin D has many other functions. Following the finding of receptor in immune cells, this group also discovered that the vitamin D hormone suppresses autoimmune diseases such as EAE (a model of human multiple sclerosis), Lupus erythematosis, and Type I diabetes. The final chapter of the vitamin D portion of this story is the development of selective analogs that are targeted to osteoblasts of bone, giving great potential for the development of a new drug for the treatment of osteoporosis. The DeLuca group isolated and identified all-trans-retinoic acid as a bonafide metabolite of vitamin A in carrying out its functions. Furthermore, they isolated and identified several metabolites of all-trans-retinoic acid, and have provided information on the role of retinol in female reproduction, highlighting it necessity at day 10 of gestation. A new metabolite of retinol was discovered and identified as a thiophene derivative whose function remains unknown. Professor Suttie's group was the leader in demonstrating that vitamin K functions to cause production of gamma carboxyglutamic residues in the clotting factors essentially elucidating the molecular mechanism of vitamin K in causing blood clotting. They further determined how Warfarin antagonizes the action of vitamin K. Their work continued on other functions of vitamin K in the gamma carboxylation of such important bone proteins as osteocalcin. This project has been funded for at least 30 years by the NIH and must be viewed as one of the most successful in the NIH history as well as in the Wisconsin Experiment Station records.

Impacts
The metabolically active form of vitamin D and its precursor were isolated, identified and synthesized along with many analogs resulting in 7 major therapies for bone disease world-wide. The vitamin D endocrine system was delineated and the mechanism of action of 1,25-dihydroxyvitamin D3 was elucidated proving valuable information for therapy. The role of vitamin K in causing gamma carboxylation of glutamate residues of clotting proteins and how the anticoagulant coumadin functions was discovered allowing a more thorough understanding of blood-clotting mechanisms.

Publications

• Veldman, C.M., Cantorna, M.T. and DeLuca, H.F. 2000. Expression of 1,25-dihydroxyvitamin D3 receptor in the immune system. Arch. Biochem. Biophys. 374:334-338.
• Cantorna, M.T., Humpal-Winter, J. and DeLuca, H.F. 2000. In vivo upregulation of interleukin-4 is one mechanism underlying the immunoregulatory effects of 1,25-dihydroxyvitamin D3. Arch. Biochem. Biophys. 377:135-138.
• Jehan, F. and DeLuca, H.F. 2000. The mouse vitamin D receptor is mainly expressed through an Sp1-driven promoter in vivo. Arch. Biochem. Biophys. 377:273-283.
• Kimmel-Jehan, C. and DeLuca, H.F. 2000. Cloning of the mouse 25-hydroxyvitamin D3-1alpha-hydroxylase (CYP1alpha) gene. Biochim. Biophys. Acta 1475:109-113.
• Brenza, H.L. and DeLuca, H.F. 2000. Regulation of 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression by parathyroid hormone and 1,25-dihydroxyvitamin D3. Arch. Biochem. Biophys. 381:143-152.
• Binkley, N.C., Krueger, D.C., Engelke, J.A., Foley, A.L. and Suttie, J.W. 2001. Vitamin K supplementation reduces serum concentrations of under-gamma-carboxylated osteocalcin in healthy adults. Am. J. Clin. Nutr. 72:1523-1528.
• Kumar, D., Greer, F.R., Super, D.M., Suttie, J. W. and Moore, J.J. 2001. Vitamin K status of premature infants: Implications for current recommendations. Pediatrics 108:1117-1122.

Progress 01/01/00 to 12/31/00

Outputs
Dr. S. Kato provided for us breeding pairs of vitamin D receptor-less mice. Using these animals, we have demonstrated that vitamin D deficiency and the vitamin D receptor-less mice produce identical phenotypes, namely a failure of support of serum calcium and a failure of mineralization of the skeleton. No evidence has been obtained suggesting other routes of vitamin D action except through its nuclear receptor. These receptor-less mice are unable to metabolize the hormone, 1,25-dihydroxyvitamin D3, because of the absence of the 24-hydroxylase enzyme. The 24-hydroxylase is induced by the vitamin D receptor when it is liganded. Thus, receptor-less mice can neither respond to nor destroy the hormonal form of vitamin D. Receptor-less female rats are fully capable of reproduction and embryonic development is approximately normal. These results are identical with our experience in vitamin D deficiency. Experiments were also conducted to demonstrate that there is no 26-hydroxylase for 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3, illustrating that the path of metabolism to excretion products of vitamin D is through the 24-hydroxylase enzyme that converts 1,25-dihydroxyvitamin D3 to calcitroic acid.

Impacts
Our results illustrate that the function of vitamin D is exclusively through the nuclear vitamin D receptor and that this genomic system is also responsible for the ultimate metabolism of the hormone to excretion products.

Publications

• Endres, B., Kato, S. and DeLuca, H.F. 2000. Metabolism of 1a,25-dihydroxyvitamin D3 in vitamin D receptor-ablated mice in vivo. Biochemistry 39: 2123-2129.

Progress 01/01/99 to 12/31/99

Outputs
We have completed chemical syntheses of a series of analogs of 1a,25-dihydroxyvitamin D3 that possess marked selectivity of action for bone and with the highest biological activity yet observed in my laboratory. The most active of these compounds is 2a-methyl-19-nor-20S-1a,25-dihydroxyvitamin D3. Almost identical activity was obtained with 2-methylene-19-nor-20S-1a,25-dihydroxyvitamin D3. The methylene derivative has been shown to be 100 times more active than 1a,25-dihydroxyvitamin D3 in the mobilization of calcium from bone. Given at a level of 32 pmol twice a week, it not only reversed the osteoporosis caused by ovariectomy in old female rats but actually increased the bone mass above that found in the sham-operated controls. These compounds are between 10-100 times more active than 1a,25-dihydroxyvitamin D3 in causing differentiation of the promyelocytes. These compounds appear to be the most effective of all analogs we have seen for the treatment of bone loss caused by depletion of estrogen. The promoter for the mouse and rat vitamin D receptor has been successfully cloned. This promoter is driven by 4 Sp1 sites at about 4 base pairs upstream from the start site. The promoter does not possess a TATA box or a CAAT box. The full structure of the mouse vitamin D receptor gene has been deduced, and it has clearly been shown that a single promoter is responsible for production of the vitamin D receptor in the mouse.

Impacts
A new family of 1,25-dihydroxyvitamin D analogs has been discovered that markedly increase bone mass of aged ovariectomized female rats. They appear to be ideal for the treatment of osteoporosis because of their anabolic nature in bone.

Publications

• Darwish, H.M. and DeLuca, H.F. 1999. Identification of a transcription factor that binds to the promoter region of the human parathyroid hormone gene. Arch. Biochem. Biophys. 365:123-130.
• Yamamoto, K., Ooizumi, H., K. Umesono, K Verstuyf, Bouillon, R, DeLuca, H.F., Shinki, T., Suda, T. and Yamada S. 1999. Three-dimensional structure-function relationship of vitamin D: Side chain location and various activities. Bioorg. Med. Chem. Lett. 9:1041-1046.
• Kimmel-Jehan, C., Darwish, H.M, Strugnell, S.A., Jehan, F., Wiefling, B. and DeLuca, H.F. 1999. DNA bending is induced by binding of vitamin D receptor-retinoid X receptor heterodimers to vitamin D response elements. J. Cell. Biochem. 74:220-228.
• McCary, L.C., Staun, M. and DeLuca, H.F. 1999. A characterization of vitamin D-independent intestinal calcium absorption in the osteopetrotic (op/op) mouse. Arch. Biochem. Biophys. 368:429-256.
• Shinki, T., Ueno, Y., DeLuca, H.F. and Suda, T. 1999. Calcitonin is a major regulator for the expression of renal 25-hydroxyvitamin D3-1a-hydroxylase gene in normocalcemic rats. Proc. Natl. Acad. Sci. USA 96:8253-8258.
• Cantorna, M.T., Humpal-Winter, J. and DeLuca, H.F. 1999. Dietary calcium is a major factor in 1,25-dihydroxycholecalciferol suppression of experimental autoimmune encephalomyelitis in mice. J. Nutr. 129:1966-1971.

Progress 01/01/98 to 12/31/98

Outputs
During the past year we have successfully demonstrated that 1,25-(OH)2D3 and a number of synthetic analogs have a marked ability to prevent the rejection of allographic transplants, both in non-vascularized and vascularized mouse and rat models. The results illustrate that these vitamin D compounds are superior to cyclosporin in that the transplant survival is markedly prolonged above that possible with cyclosporin. The two agents act synergistically. This will lead to the development of the vitamin D compounds to support transplantation of organs in the organ transplantation group in the Department of Surgery. We have continued to exploit the cloning of the 25-hydroxyvitamin D-1a-hydroxylase and have been able to isolate the gene for that enzyme. By splicing the promoter of that gene in a Luciferase reporter system, we can now demonstrate in AOK B-50 cells that parathyroid hormone has a marked stimulatory activity. We have mapped this activity to 150 bases of the 3' end of the reporter. The site appears to be overlapping with the TATA box that is required for expression of the promoter. We have identified a new metabolite of vitamin A as a dihydrothiophene derivative derived from the terminal piece of the retinol molecule.

Impacts
The use of 1,25-(OH)2D3 analogs could revolutionize the immunosuppression aspect of transplantation and prolong the health and success of organ transplantation in man.

Publications

• Strugnell, S.A., Wiefling, B.A., and DeLuca, H.F. 1997. A modified pGEX vector with a C-terminal histidine tag: Recombinant double-tagged protein obtained in greater yield and purity. Analyt. Biochem. 254:147-149.
• McCary, L.C., Smith, C.M., and DeLuca, H.F. 1997. Hypophosphatemia and the development of rickets in osteopetrotic (op/op) mice. J. Bone Min. Res. 12:1944-1951.
• Cantorna, M.T., Hayes, C.E., and DeLuca, H.F. 1998. 1,25-Dihydroxycholecalciferol inhibits the progression of arthritis in murine models of Human Arthritis. J. Nutr. 128:68-72.
• Brenza, H.L., Kimmel-Jehan, C., Jehan, F., Shinki, T., Wakino, S., Anazawa, H., Suda, T., and DeLuca, H.F. 1998. Parathyroid hormone activation of the 25-hydroxyvitamin D3-1a-hydroxylase gene promoter. Proc. Natl. Acad. Sci. USA 95:1387-1391.
• Jehan, F., Ismail, R., Hanson, K., and DeLuca, H.F. 1998. Cloning and expression of the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA. Biochim. Biophys. Acta 1395:259-265.
• Jia, X., Sicinski, R.R., Wellik, D.M., Tadikona, P., Schnoes, H.K., and DeLuca, H.F. 1998. Identification of a new all-trans-retinol metabolite produced through a new retinol metabolic pathway. Biochemistry 37:5974-5980.

Progress 01/01/97 to 12/31/97

Outputs
Using the cDNA clone of the rat 25-hydroxyvitamin D-1a-hydroxylase, we have been able to obtain the full length cDNA of the mouse 25-(OH)2D-1a-OHase. With this as a probe, we have now obtained a genomic clone encoding the 1a-OHase. From this we have isolated a promoter and have demonstrated its ability to initiate transcription of a reporter gene. We have found that this sequence contains a PTH-sensitive responsive system and have narrowed it to the first 125 bases upstream from the transcriptional start site. We have synthesized two new important analogs of 1,25-(OH)2D3: 2-methylene-19-nor-1,25-(OH)2D3 and 2a-methyl-19-nor-1,25-(OH)2D3. These compounds are 10-1,000 times more potent than 1,25-(OH)2D3 itself in mobilizing calcium from bone and in stimulating differentiation of HL-60 cells. In vivo these compounds appear to be specific for action on the osteoblast. We have isolated in pure form a new metabolite of retinol (vitamin A) and have determined its structure. It contains a 5-member ring including 1 sulfur atom. We have confirmed the structure by chemical synthesis. This compound appears to be effective in carrying out the function of vitamin A in female reproduction.

Impacts
(N/A)

Publications

• Elaroussi, M.A. and DeLuca, H.F. 1997. CAM-1. A new member to the Astacin family of metalloendopeptidases regulated by vitamin D from the chorioallantoic membrane of quail. In The Astacins. Structure and Function of a New Protein Family. Zwilling, R. and W. Stocker, eds., pp. 275-289. Verlag Dr. Kovac, Hamburg, Germany.
• McCary, L.C., Smith, S.M. and DeLuca, H.F. 1997. Rickets in osteopetrotic, op-op, mice results from hypophosphatemia and not from 1,25-OH2D3 resistance. J. Bone Miner. Res. 12:1944-1951.
• Tadikonda, P.K. and DeLuca, H.F. 1997. Preparation of radiolabeled 9-cis- and 11-trans-retinoids. In Methods in Enzymology. Vitamins and Coenzymes. D.B. McCormick, J.W. Suttie, and C. Wagner, eds., Volume 282, Chapter 10, pp. 108-117. Academic Press, New York.
• Tadikonda, P.K., Lacy, J.M., Rigdon, M.G. and DeLuca, H.F. 1997. Synthesis of 9-cis-retinoic acid and C-20 3H3C-9-cis-retinoic acid with high specific activity. J. Label. Compounds and Radiopharm. XXXIX:1-10.
• Wellik, D.M., Norback, D.H. and DeLuca, H.F. 1997. Retinol is specifically required during midgestation for neonatal survival. Am. J. Physiol. 272:E25-E29.
• Lu, Z., Hanson, K. and DeLuca, H.F. 1997. Cloning and the origin of the two forms of chicken vitamin D receptor. Arch. Biochem. Biophys. 339:99-106.
• Kimmel-Jehan, C., Jehan, F. and DeLuca, H.F. 1997. Salt concentration determines 1,25-dihydroxyvitamin D3 dependency of vitamin D receptor--retinoid X receptor--vitamin D-responsive element complex formation. Arch. Biochem. Biophys. 341:75-80.
• Strugnell, S.A. and DeLuca, H.F. 1997. Minireview. The vitamin D receptor--structure and transcriptional activation. Proc. Soc. Exp. Biol. Med. 215:223-228.
• DeLuca, H.F. 1997. Historical overview. In: Vitamin D. Feldman, D., F.H. Glorieux, and J.W. Pike, eds., Chapter 1, pp. 3-11. Academic Press, San Diego, CA.
• Jehan, F. and DeLuca, H.F. 1997. Cloning and characterization of the mouse vitamin D receptor promoter. Proc. Natl. Acad. Sci. USA 94:10138-10143.
• McCary, L.C., Smith, C.M. and DeLuca, H.F. 1997. Hypophosphatemia and the development of rickets in osteopetrotic, op-op, mice. J. Bone Min. Res. 12: 1944-1951.
• Beckman, M.J. and DeLuca, H.F. 1997. Assay of 1,25-dihydroxyvitamin D3 from serum samples: use of receptor-binding or enzyme-coupled reporter analysis. In Methods in Enzymology. Vitamins and Coenzymes. D.B. McCormick, J.W. Suttie, and C. Wagner, eds., Volume 282, Chapter 15, pp. 164-174. Academic Press, New York.
• Beckman, M.J. and DeLuca, H.F. 1997. Assay of 25-hydroxyvitamin D 1a-hydroxylase and 24-hydroxylase. In Methods in Enzymology. Vitamins and Coenzymes. D.B. McCormick, J.W. Suttie, and C. Wagner, eds., Volume 282, Chapter 18, pp. 200-213. Academic Press, New York.
• Strugnell, S.A., Wiefling, B.A., and DeLuca, H.F. 1997. A modified pGEX vector with a C-terminal histidine tag: recombinant double-tagged protein obtained in greater yield and purity. Analyt. Biochem. 254:147-149.
• DeLuca, H.F. 1997. 1,25-Dihydroxyvitamin D3 in the pathogenesis and treatment of osteoporosis. Osteoporosis Int. 7:S24-S29.
• DeLuca, H.F. 1997. The genetics and biology of vitamin D. In: Principles of Medical Biology, vol. 10B. Bittar, E.E. and N. Bittar, eds., JAI Press, Inc., Greenwich, CT. Chapter 29, pp. 617-641.

Progress 01/01/95 to 12/30/95

Outputs
The 25-hydroxyvitamin D 1-hydroxylase responsible for activation of vitamin D toits hormonal form has been highly purified and an amino acid sequence has been obtained on the putative enzyme. Murine experimental encephalomyelitis has been induced in BL10 mice by injection of myelin basic protein. This disease is considered a model of human multiple sclerosis and is completely prevented by the administration of 1,25- dihydroxyvitamin D3 (1,25-(OH)2D3). Furthermore, the administration of 1,25-(OH)2D3 at any stage stops progression of the disease and causes improvement to normal. These results demonstrate that 1,25-(OH)2D3 and its analogs may be used to treat human multiple sclerosis. Work has continued on isolating factors required for the vitamin D receptor (VDR) to form a transcription complex on vitamin D responsive elements in responsive genes. It has been conclusively shown that the RXR series of proteins and at least one other protein is needed to cause the VDR to form a complex on the vitamin D response element. We have identified ranscription factor TFIIB as one of the proteins required for 1,25-(OH)2D3-induced transcription of a target gene. The VDR has now been expressed in two bacterial expression systems, both as a His-tag protein and as a fusion protein with glutathione transferase. This expression has resulted in the preparation of large amounts of receptor and its parts in pure form for determination of their 3-D structures.

Impacts
(N/A)

Publications

• Zierold, C., Darwish, H.M., and DeLuca, H.F. 1995. Two vitamin D response elements function in the rat 1,25-dihydroxyvitamin D 24-hydroxylase promoter. J.Biol. Chem. 270:1675-1678.
• Ettinger, R.A. and DeLuca, H.F. 1995. The vitamin D3 hydroxylase-associated protein is a propionamide-metabolizing amidase enzyme. Arch. Biochem. Biophys. 316:14-19.
• Munder, M., Herzberg, I.M., Zierold, C., Moss, V.E., Hanson, K., Clagett-Dame, M., and DeLuca, H.F. 1995. Identification of the porcine intestinal accessory factor that enables DNA sequence recognition by vitamin D receptor. Proc. Natl. Aca
• Chen, K.-S. and DeLuca, H.F. 1995. Cloning of the human 1,25-dihydroxyvitamin D-3 24- hydroxylase gene promoter and identification of two vitamin D-responsive elements. Biochim. Biophys. Acta 1263:1-9.
• McCary, L.C. and DeLuca, H.F. 1995. Osteopetrotic (op/op) mice are unable to maintain serum calcium levels despite hyperabsorption of calcium. Endocrinology 137:1049-1056.
• Wellik, D.M. and DeLuca, H.F. 1995. Retinol in addition to retinoic acid is required for successful gestation in vitamin A-deficient rats. Biol. Rep. 53:1392-1397.
• Perlman, K.L., Sicinski, R.R., Darwish, H.M., and DeLuca, H.F. 1995. Synthesis.

Progress 01/01/93 to 12/30/93

Outputs
We have been able to produce mg amounts of the 1,25-dihydroxyvitamin D(subscript3) (1,25-(OH)(subscript 2)D(subscript 3)) receptor using the baculovirus expression system. By immobilizing monoclonal antibodies directed against the receptor, we can in a single-step process produce pure receptor suitable for crystallography and for nuclear magnetic resonance spectroscopy to determine its three-dimensional structure. Using gel shift analysis, as well as reporter gene analysis, we have demonstrated the existence of and elucidated a vitamin D-response element in the promoter region of the rat 25-OH-D-24-hydroxylase gene. In addition to this response element located on base -238 to -262 (from transcription initiation site), another vitamin D response element was found in the same promoter at -154 to -134 bases prior to start site. Two unique proteins have been cloned as a result of our recombinant work. One is a vitamin D-dependent protein of unknown function which appears in HL-60 cells following stimulation by 1,25-(OH)(subscript 2)D(subscript 3). Although we have determined the entire amino acid sequence, we have not determined its function. Another protein was cloned as a result of our work on the 24-hydroxylase. This protein is an amidase found exclusively in the kidney and mitochondria. It is not regulated by vitamin D. We have chemically synthesized 19-nor-1(alpha),2(alpha),25-(OH)(subscript 2)D(subscript 3) and a 19-nor-1(alpha),2(beta)-hydroxy-25-(OH)(subscript 2)D(subscript 3).

Impacts
(N/A)

Publications

Progress 01/01/92 to 12/30/92

Outputs
Using the previously reported recombinantly produced 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) receptor, we have demonstrated that the binding to its response element in target genes requires the presence of another nucleoprotein, the nuclear accessory protein. Similarly, if porcine intestinal receptor is purified to homogeneity, it also requires the accessory protein for binding to its response element. This response element has been purified and has a molecular weight in the range of 60,000. A Vitamin D-response element has been located in the calcium binding protein found in rat intestine. The response element has been thoroughly characterized and joins those previously reported. Activators of protein kinase A, which produce phosphorylation of the Vitamin D receptor, will activate this receptor transcriptively without ligand. Furthermore, inhibitors of protein kinase A will block Vitamin D-induced response in a reporter gene system. These results provide important evidence that phosphorylation of the Vitamin D receptor is an essential step in activation of the receptor. The human 25-OH-D-24-hydroxylase has been cloned and its entire sequence determined. This enzyme has been over-expressed in baculovirus and in cos cells, demonstrating that we have, in fact, isolated the cDNA for the 24-hydoxylase. In the area of Vitamin A, we have successfully expressed the gamma-retinoic acid receptor in baculovirus and have markedly purified it.

Impacts
(N/A)

Publications

Progress 01/01/91 to 12/30/91

Outputs
We have successfully over-expressed the Vitamin D receptor in a baculovirus expression system yielding large amounts, i.e. 10% of the cellular protein. The recombinant receptordoes not bind the Vitamin D-response elements of target genes until another (accessory) protein from porcine nuclear extract is added. The accessory protein is found in target tissues but not in non-target tissues. A Vitamin D response element has now been located at approximately 500 bases upstream from the start site on the calbindin D-9k rat gene. This response element has two repeat sequences, separated3 bases. Antibodies raised to the 24-hydroxylase of 1,25-(OH)(2)D(3) have been obtained and used to immunopurify the 24-hydroxylase and 1alpha-hydroxylase from chicken kidney mitochondria. The two proteins are homologus, but are distinct by virtue of a different amino acid composition and by virtue of slightly different N-terminal sequences. A series of new 1,25-(OH)(2)D analogs known as 19-nor-1alpha, 25-(OH)(2)D compounds have been synthesized, most of them showing markedly reduced calcemic activity while retaining the ability to differentiate HL-60 cells in culture. A new method for synthesizing the 19-nor series of compounds has been devised which involves total synthesis starting from quinic acid. The alpha and beta receptors for all-trans-retinoicacid have been demonstrated for neuroblastoma cells.

Impacts
(N/A)

Publications

Progress 01/01/90 to 12/30/90

Outputs
Major emphasis has been placed on the molecular mechanism of action of 1,25-(OH)(subscript 2 )D(subscript 3 ), in the small intestine and in osteoblasts. 1,25-Dihydroxyvitamin D(subscript 3 ) responsive elements in the promoter region of the rat osteocalcin gene and the mouse osteopontin gene have been identified using chloramphenicol acetyltransferase reporter gene system and gel shifts. We have also identified an estrogen-response element at the end of the first exon and the beginning of the first intron of the 9K calcium binding protein gene. In the uterus, both in vivo and in vitro techniques have demonstrated that estrogen is the major regulatory agent. The 1,25-(OH)(subscript 2 )D(subscript 3 ) receptor is rapidly phosphorylated in vivo after it has bound 1,25-(OH)(subscript 2 )D(subscript 3 ) before it can stimulate transcription. This phosphorylation occurs on serines in the 1,25-(OH)(subscript 2 )D(subscript 3 ) binding region of the receptor. New analogs of 1,25-(OH)(subscript 2 )D(subscript 3 ) have been prepared that restore bone lost in female rats as a result of oophorectomy. One of them, 24-epi-1(alpha),25-(OH)(subscript 2 )D(subscript 3 ) appears very promising as a treatment for postmenopausal osteoporosis. A peptide has been synthesized which is derived from the reported amino acid sequence of the gamma receptor for retinoic acid.

Impacts
(N/A)

Publications

Progress 01/01/89 to 12/30/89

Outputs
Research during the past year has continued to focus on the molecular mechanism of action of the vitamin D hormone. Following the characterization/sequencing of full length cDNA clones of the genes for 1,25-(OH)(2)D(3) receptor and calcium binding protein in rat intestine as reported last year, we have demonstrated that expression of the 1,25-(OH)(2)D(3) receptor gene is tissue specific, and that, in accord with our earlier work, gene expression in neonatal development occurs after 18-21 days of age. In further studies on the genes regulated by 1,25-(OH)(2)D(3), we have identified six potential candidates in rat intestine, one of which is the gene for 1,25-(OH)(2)D(3) receptor protein itself. It was also shown that 1,25-(OH)(2)D(3) stimulates the rapid incorporation of phosphorus into receptor protein, implicating receptor phosphorylation as a key event in receptor function. An immunoprecipitation sandwich assay for total 1,25-(OH)(2)D(3) receptor has been developed; the assay is sensitive to 1-2 pg of receptor protein. Similarly, a highly sensitive, rapid, quantitative assay for 1,25-(OH)(2)D(3), involving immunoprecipitation with biotinylated antibody & avidin, has been devised; the assay allows triplicate 1,25-(OH)(2)D(3) determinations on as little as 50-200 mu l of blood.

Impacts
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Publications

Progress 01/01/88 to 12/30/88

Outputs
The full length cDNA encoding for the entire 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) rat receptor has been cloned, and its sequence determined. The deduced amino acid sequence agrees exactly with partial amino acid sequence obtained on pure isolated porcine intestinal 1,25-(OH)(2)D(3) receptor. The latter was obtained by means of an immunoaffinity column in which a monoclonal antibody against the porcine receptor was immobilized on Sepharose CL4B. The structure of the rat receptor is largely homologous to the reported human receptor structure in the DNA binding region and the ligand binding region. Continued attempts to demonstrate the presence of a 1,25-(OH)(2)D(3) responsive element in the calcium binding protein gene isolated from the rat has not met with success. By means of reporter genes we have probed the 5' and 3' ends of the calcium binding protein gene and the introns but none of these regions appear to have a sequence that results in stimulation of transcription of the CAT reporter system. We have developed a new chemical synthesis in which we can rapidly alter the side chain structure of 1,25-(OH)(2)D(3). We have prepared a large number of side chain analogs using this approach. Testing of some of them have revealed that by elongating the side chain in the 24-position, we can construct a compound that is active in causing differentiation but which has no activity in mobilizing calcium.

Impacts
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Publications

Progress 01/01/87 to 12/30/87

Outputs
The cDNA encoding for the rat intestinal receptor for 1,25-dihydroxyvitamin D(3)(1,25-(OH)(2)D(3)) has been isolated and sequenced. The protein produced from this cDNA binds specifically 1,25-(OH)(2)D(3) and reacts with the monoclonal antibodies directed to the porcine intestinal receptor for 1,25-(OH)(2)D(3). The receptor has also been isolated in pure form by immunoaffinity chromatography and a chemical sequence obtained on 4 peptides from proteolytic digests. The sequences obtained there agree with the sequences predicted by the cDNA clone. Full length cDNA for intestinal calcium binding protein from rat has also been obtained and the sequence completed. The entire calcium binding protein gene has also been isolated from the rat genomic library. It has been sequenced and the regulatory region to which the 1,25-(OH)(2)D(3) receptor plus ligand bind has been deduced. A series of homologs of 1,25-(OH)(2)D(3) has been synthesized chemically and shown to have specific action in suppressing growth and causing differentiation of malignant leukemia cells in culture, while having diminished activity on calcium metabolism in animals. These compounds are of great interest as possible therapeutic agents for malignancy. The metabolities of retinoic acid have been isolated from tissues of rats given a physiological dose of retinoic acid. They have been shown to be biologically inactive except for retinoyl-beta-glucuronide and 13-cis-retinoic acid.

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Publications

Progress 01/01/86 to 12/30/86

Outputs
Using monoclonal antibodies directed on the 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) receptor, it can be demonstrated in the small intestine that the mammalian receptor has a molecular weight of 55,000 but demonstrates charge heterogeneity. Three species are found; one with a pI of 5.9, another with a pI of 6.1, and still another that does not enter the gel from the electrofocusing direction. Highly purified receptor can now be obtained with immunoaffinity resins synthesized with the monoclonal antibodies together with a lambda GT-11 cDNA library from rat intestinal mRNA. We have isolated at least one clone of cDNA coding for a portion of the receptor protein. This has been sequenced and likely represents the DNA binding site. A full-length cDNA coding for rat intestinal calcium binding protein has been isolated and sequenced. We have also isolated the rat calcium binding protein gene within a 4 kb genomic fragment. The 25-OH-D-24-hydroxylase has been solubilized and shown it to be a cytochrome P-450 three-component mixed function monooxygenase. We have developed new rapid assays for measurement of the 25-OH-D-1 alpha-hydroxylase and 24-hydroxylase. 1 alpha-tritium-25-OH-D synthesis was carried out to accomplish one of the above assays.

Impacts
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Publications

Progress 01/01/85 to 12/30/85

Outputs
The receptor for 1,25-(OH)(2)D(3) has been isolated in excellent yield at a purity of approximately 50% from hog intestinal mucosa. It has been identified as a protein of 55,000 molecular weight and a pI of 6.1. 24 monoclonal antibodies have been generated to this receptor, 17 of which have been well characterized. 8 of these react in an immunoblot on SDS gel electrophoresed receptor. All but 2 crossreact with receptor from a variety of species, whereas 2 are specific for the pig. The calcium binding protein gene has been isolated from a genomic library and is being studied for specific binding sites to the 1,25-(OH)(2)D(3) receptor. Photoaffinity and affinity labels have been prepared for the receptor and the vitamin D binding protein, and homologs of the vitamin D hormone have been prepared that preferentially stimulate myeloid leukemia differentiation in culture versus calcium mobilization in vivo. Appropriate derivatives have been prepared that show that the 13-cis and all-trans-retinoic acid are active in vivo. A heavily derivatized analog of retinoic acid is more active than retinoic acid, illustrating that it is unlikely that retinoic acid is metabolically modified before function. Retinoic acid is converted to retinoyl co-A before retinoic acid is transferred to an alcoholic acceptor, namely ethyl alcohol.

Impacts
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Publications

Progress 01/01/84 to 12/30/84

Outputs
Porcine intestinal 1,25-(OH)(2)D receptor has been purified to 50% purity and used to generate 24 monoclonal antibodies. These antibodies cross-reaction with chicken, rat, and human intestinal receptor. Chemical synthesis of 25-OH-D(2) and 1,25-(OH)(2)D(2) and their 24-epimers in stable and radioactive form have been completed. Chemical synthesis of 24R,25-(OH)(2)-(26,27-H)D(3) has also been completed and used for study of metabolism. Two-dimensional electrophoresis coupled with computer-driven analysis of films generated from tritiated leucine-labeled proteins and C-labeled proteins have been developed to examine genes expressed in response to 1,25-(OH)(2)D(3). Intestinal calcium binding protein synthesis precedes intestinal calcium transport and at least two other proteins have been detected that are responsive to 1,25-(OH)(2)D(3). 1,25-(OH)(2)D(3) is not transferred from hen to egg in sufficient quantity to support calcium mobilization of egg shell for chick embryo during the terminal stages of incubation. These chicks becomes vitamin D deficient, having hypocalcemia, extreme hyperphosphatemia, and they fail to hatch. Retinoic acid modified with two fluoro groups on C-4 or extensive modification that prevents metabolism does not diminish but rather increases biological activity, suggesting that if metabolic alteration of retinoic acid must occur before function, it must be on the terminal carboxyl group or the gem methyls.

Impacts
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Publications

Progress 01/01/83 to 12/30/83

Outputs
Methods for studying gene expression in response to 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) have been developed using two-dimensional gel electrophoresis and electrofocusing plus double-label protein (MW 27,000) makes its appearance in embryonic chick intestine prior to initiation of calcium transport in response to 1,25-(OH)(2)D(3). We have recently conclusively demonstrated that vitamin D and its metabolites do not function directly on the mineralization process or on the synthesis of organic matrix of bone. The function of vitamin D and its metabolites in mineralizing bone is striclty by elevation of plasma calcium and phosphorus concentrations to the normal range. The work also demonstrated that vitamin D is involved in the bone resorption process since in its absence, bone accumulates in excessive amounts if calcium and phosphorus infusions are continued. Chemical synthesis of 25-hydroxyvitamin D(2) (25-OH-D(2)) and its 24-epimer has been completed. Similarly, synthesis of 1,25-(OH)(2)D(2) and its 24-epimer has also been completed. 1,25-(OH(2))D(3) is responsible for the developmental changes that occur in chorioallantoic membrane. However, 1,25-(OH)(2)D(3) is likely not transferred from hen to egg. It is, therefore, required that hens receive either vitamin D or 25-OH-D in order to transfer sufficient amounts to the egg so that the egg will not be vitamin D deficient.

Impacts
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Publications

Progress 01/01/82 to 12/30/82

Outputs
Work has continued on the purification on chick and pig intestinal cytosol receptor for 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)). Two independent methods of isolating the chick protein have been devised and have resulted in pure receptor as determined by gel electrophoresis. It is a single polypeptide with a molecular weight of 63,000 and has approximately 4 sufhydryl groups. The receptor is found in rat pup bone prior to parturition, whereas in the small intestine it does not appear until just prior to weaning. At this time the intestine becomes sensitive to vitamin D compounds and intestinal calcium transport begins. Two-dimensional gel electrophoreses, double-labeled techniques, autoradiography, fluorography, and computer analyses have been used to study 1,25-(OH)(2)D(3)-induced proteins. Calcium binding protein appears within a half-hour post-treatment of chick embryonic intestine with 1,25-(OH)(2)D(3) and long before calcium uptake begins. 24,24-F(2)-25-OH-D(3) and 26,26,26,27, 27,27, -F(6)-25-OH-D(3) have been used to demonstrate that 24-hydroxylation, 26,23-lactone formation, and 26-hydroxylation of vitamin D compounds are not involved in the target organ responses to vitamin D. Furthermore, animals grown for two generations in the absence of 24-hydroxylation show no distinguishable lesion.

Impacts
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Publications

Progress 01/01/81 to 12/30/81

Outputs
The receptor protein for 1,25-(OH)(2)D(3) has been isolated in pure form from intestinal mucosa of chicks. This protein has a molecular weight of 67,000 and is a single polypeptide. This receptor is absent in the intestines of suckling young rats that accounts for their lack of responsiveness to vitamin D and its hormone. Furthermore, this receptor is absent in vitamin D-dependency rickets Type II, a genetic disorder, explaining the basis of this disease. These two results illustrate that the vitamin D hormone must interact with the receptor protein before it elicits an increase in intestinal calcium and phosphate transport. 23S, 25-(OH)(2)D(3) has been isolated as an important new metabolite of vitamin D. It has been shown to be a precursor for the biogenesis of 25-OH-D(3)-26,23-lactone. Both metabolites of vitamin D are devoid of biological activity. This system is found entirely in the kidney. Retinoic acid has shown to be converted to retinoyl-Beta-glucuronide both in cis and trans forms in vivo in target tissues. This compound is at least as active as retinoic acid and in some systems appears more active. It is a possible metabolically active form of vitamin A. 5,6-Epoxyretinoic acid has been shown to be essentially devoid of biological activity and, furthermore, the enzyme responsible for its production can be blocked with DPPD.

Impacts
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Publications

Progress 01/01/80 to 12/30/80

Outputs
Increased production of the 1,25(OH)(2)O(3) during pregnancy and lactation has been demonstrated. However, Vitamin D deficient rats are able to conceive, carry young to term, and lactate them to approximately normal development, illustrating that although Vitamin D and its hormone are helpful during pregnancy and lactation, they are not necessary. These results illustrate that some other humoral factor is secreted during pregnancy and lactation in mammals responsible for mobilizing calcium from bone and small intestine. The Vitamin D deficient suckling pups absorb calcium normally from milk derived from Vitamin D-deficient mothers. Calcium transport in intestine of suckling pups during the first 14 days of lactation is not dependent upon Vitamin D, and in fact, the intestine is non-responsive to Vitamin D and its hormone. It is only consumption of non-milk food that the intestines of pups becomes responsive to 1,25(OH)(2)O(3). Milk contains very little actual Vitamin D activity and can be accounted for entirely by the known metabolites of Vitamin D. There is no evidence for the claimed super-active water soluble form of Vitamin D in milk. Retinoyl-Beta-glucuronide has been isolated from the bile of rats given physiological or pharmacological doses of retinoic acid. It accounts for only ten percent of the biliary excretory radioactivity derived from retinoiac acid, unlike previous reports. It is unlikely to represent the major excretory form of retinoiac acid but its biological role remains unknown.

Impacts
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Publications

Progress 01/01/79 to 12/30/79

Outputs
A direct chemical synthesis of high specific activity 26,27-tritium labelled 1,25-dihydroxyvitamin D(3) has been achieved giving a compound possessing 160 Ci/mmole radioactivity. With this compound, target organs of 1,25-dihydroxyvitamin D(3) have been identified by nuclear localization using frozen section autoradiography. No nuclear localization has been found in muscle, submucosa of intestine, all other cells of the kidney, spleen, liver, nerve, and fibroblasts. A study of skin has revealed that 1,25-dihydroxyvitamin D(3) markedly increased the accumulation of 7-dehydrocholesterol in that organ, suggesting a positive feedback control of vitamin D biogenesis in the skin by 1,25-dihydroxyvitamin D(3). Biological activity studies have been completed on calcitroic acid, the major metabolite of 1,25-dihydroxyvitamin D(3), revealing it to be of weak biological acitivty, suggesting it to be a degradation product. A new metabolite of 24,25-dihydroxyvitamin D(3) has been isolated and identified as the 25,26,27-trinor-vitamin D(3), 24-carboxylic acid. Its diminished biological activity suggests it to be a primarily inactivation product.

Impacts
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Publications

Progress 01/01/78 to 12/30/78

Outputs
A major metabolite of the native hormone 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) has been isolated in pure form and positively identified as la-hydroxy-24,25,26,27-tetranorvitamin D(3)23-carboxycyclic acid, (calcitroic acid). Another new metabolite of 25-hydroxyvitamin D(3) (25-OH-D(3)) has been isolated from the plasma of chicks either given physiologic or massive amounts of vitamin D. A number of fluoro analogs of 1,25-(OH)(2)D(3) have been prepared and these metabolites have been used to show that blockage of the 25 position does not result in the inactivity. New chemical syntheses of high specific activity 25-OH-D(3) and 1,25-(OH)(2)D(3) has been developed. In birds, vitamin D is necessary for upper mandible development, and the supply of 1,25-(OH)(2)D(3) to laying hens does not allow for normal development of the upper mandible. However, injection of eggs from D deficient-hens or hens supported on 1,25-(OH)(2)D(3), results in normal mandible development and thus normal hatchability. An investigation of the vitamin hydroxylases has continued with the demonstration that the 25-hydroxylase is not only a mixed-function mono-oxygenase as shown by oxygen-18 experiments, but also is a cytochrome P450 dependent reaction, as shown in inhibitor studies. A metabolite of vitamin A has been isolated in pure form from intestinal mucosa from vitamin A deficient rats given radioactive retinoic acid.

Impacts
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Publications

Progress 01/01/77 to 12/30/77

Outputs
A new method for the introduction of a hydroxyl function in the la position on the vitamin D molecule has been developed which involves the conversion of the corresponding vitamin to the 3,5-cyclo vitamin, its oxidation by selenium dioxide and subsequent reversion of the cyclo vitamin to la-hydroxyvitamin. Chemical synthesis of 1B, 25-(OH)(2)D(3) and 1B-OH-D(3) has been completed and the former demonstrated to be distinctly different from the natural product la, 25-(OH)(2)D(3). A synthesis has been developed for 3a-tritium labelled vitamin D compounds involving an iron carbonyl complex of the vitamin, oxidation to the 3-ketone and reduction with the corresponding borotritide. The synthesis of 25-aza-vitamin D(3) an anti-vitamin D compound and synthesis of 25-fluoro 24-dehydro and 25-dehydro vitamin D(3) have also been completed. X-ray crystallography has been completed on the 25-OH-D(3) monohydrate and its detailed physical, chemical arrangement of bonds have been determined. 1,24R, 25-(OH)(3)D(3) has been shown to be the natural isomer of the compound by cochromatography on high pressure liquid chromatography with synthetic 1,24R, 25-(OH)(3)D(3). Chronic adminstration of 1,2-tritiated vitamin D(3) to vitamin D deficient animals for at least a week brings about the appearance of 7 new metabolites of vitamin D(3). Development of assays for vitamin D metabolites has continued.

Impacts
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Publications

Progress 01/01/76 to 12/30/76

Outputs
It has been unequivocally demonstrated that 1,25-(OH)(2)D(3), the active form ofvitamin D, is metabolized to yield a product which has part of its side chain oxidized to carbon dioxide and water. A cytosol receptor for 1,25-(OH)(2)D(3) has unequivocally been demonstrated in the intestinal epithelium and embryonic bone of chicks and rats. A high-pressure liquid chromatographic system for the separation of all of the known metabolites and analogs of vitamin D has been developed and applied to the analysis of metabolites in the plasma. In addition, a combination of high-pressure liquid chromatography plus the specificchick intestinal receptor has permitted the development of a highly sensitive and reproducible assay for 1,25-(OH)(2)D(3). In the laying bird there is a highlevel of 25-OH-D(3)-1-hydroxylase, whereas in their male counterparts this enzyme has very low activity. It can be markedly stimulated by the injection ofestradiol and in castrate male birds, a combination of estrogen and testosteron markedly stimulate the 25- OH-D(3)-1-hydroxylase. It is potulated that 1,25-(OH)(2)D(3) plays an essential role in the mobilization of medullary bone during the formation of egg shells. Three new analogs of vitamin D have been prepared, 1alpha-hydroxyvitamin D(2), 3-deoxy-1alpha-hydroxyvitamin D(3), and 1alpha OH-pregnacalciferol. 1alpha-OH-D(2) is approximately equal to 1alpha-OH-D(3) in biological activity. The pregnacalciferol

Impacts
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Publications

Progress 01/01/75 to 12/30/75

Outputs
Tritium labeled 1x-OH-DDT has been chemically synthesized and shown to be hydroxylated on carbon 25 in the liver before it carries out its functions in intestine and bone. In the chicken some 25-hydroxylation occurs in intestine but none occurs in the intestine of rats. An iron sulfur protein called renal ferredoxin which transfers electrons from flavoprotein to the cytochrome P-450 which carries out the 1-hydroxylation reaction has been isolated from chick kidney mitochondria. This protein has a molecular weight of 12,500 and is immunologically similar to the iron sulfur protein which has been isolated from beef adrenal glands and which functions similarly in the production of steroid hormones. With 25-OH-(26,27-(14)C)DDT and 1,25-(OH)y-(26,27-(14C)DDT, a new pathway of vitamin D metabolism has been demonstrated in which 1,25-(OH)yDDT undergoes side chain oxidation to lose their 26 and 27 carbons to carbon dioxide. A protein has been isolated from rat plasma which inhibits 25-OH-DDT-1-hydroxylase obtained from chicken kidney mitochondria. This inhibitor proved to be the plasma transport protein for 25-OH-DDT. It has a molecular weight of 51,000 and an antibody to it has been prepared. 1,25-(OH)yDy, 24,25-(OH)yDy and 24-OHDy have been isolated and their structures determined chemically. The chick discriminates against all forms of vitamin Dy including 1,25-(OH)yDy. It appears that substitutions on the 24 position in thisspecies signals rapid metabolism and

Impacts
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Publications

Progress 01/01/74 to 12/30/74

Outputs
A role of 1,25(OH)(2)D(3) in phosphate metabolism independent of its role in calcium metabolism has been demonstrated. This compound stimulates a phosphate transport system in intestines and elevates serum phosphate. The rise in serum phosphate is primarily responsible for mineralization of bone while the calcium mobilization role prevents tetany. Chemical synthesis of 24,25(OH)(2)D(3) and 3deoxy lalpha hydroxy vitamin D(3) have been completed. The 3 deoxy lalpha OH-D(3) stimulates intestinal calcium absorption but does not stimulate mobilization of bone calcium or rise in serum Pi proving the phosphate and calcium systems activated by 1,25(OH)(2)D(3) are independent of each other. Clinical testing has revealed that 1,25(OH)(2)D(3) and lalpha OH-D(3) are very effective in the treatment of hypoparathyroidism, pseudohypoparathyroidism, renal osteodystrophy and certain types of vitamin D resistant rickets. The lalpha OH-D(3) has also been shown to be very effective against milk fever disease. The 25 OH-D(3)-lalpha-hydroxylase from chick kidney mitochondria has been shown to be a cytochrome OPI450 dependent reaction. It has been solubilized and partially purified. The 25-OH-D(3)-24-hydroxylase is similarly located but evidence is lacking that it depends upon a cytochrome OPI450 enzyme.

Impacts
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Publications

Progress 01/01/73 to 12/30/73

Outputs
A new metabolite of vitamin D(3) has been isolated in pure form and unequivocally identified as 1,24,25-(OH)(3)D(3). This metabolite stimulates calcium absorption but does not appreciably stimulate bone calcium mobilization at physiologic doses. It is formed from 24,25-(OH(2)D(3) and accounts for the latter's ability to support growth, serum calcium and intestinal calcium transport in vitamin D deficient rats fed normal levels of dietary calcium and phosphorus. Its biological importance is currently being evaluated. 1alpha-OH-D(3), an analog of 1,25-(OH)(2)D(3) has been chemically synthesized and its biopotency tested. It is 5 times more active than vitamin D(3) and is 1/2 as active as 1,25-(OH(2)D(3) in stimulating bone mineralization, intestinal calcium transport and bone calcium mobilization. Like 1,25-(OH(2)D(3) it is active in nephectomized rats while 25-OH-D(3) is not showing its potential in the treatment of renal osteodystrophy. Low blood phosphate as well as parathyroid hormone stimulate production of 1,25-(OH(2)D(3). It is suggested that renal cell phosphate levels determine the synthesis of 1,25-(OH)(2)D(3) andthe parathyroid hormone stimulates 1,25-(OH)(2)D(3) production by lowering renalcell level of inorganic phosphorus.

Impacts
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Publications

Progress 01/01/72 to 12/30/72

Outputs
A new metabolite of vitamin D has been isolated in pure form and its structure identified as 24,25-(OH)(2)D(3). It is formed when the synthesis of 1,25-(OH)(2)D(3) is suppressed but its function is unknown at present. The 1,25-(OH)(2)D(3) has been shown to be a metabolically active form of vitamin D in intestinal calcium transport and bone calcium mobilization. Its formation isregulated by the parathyroid glands which monitor serum calcium. In response tolow blood calcium, parathyroid hormone is secreted which then stimulates production of the 1,25-(OH)(2)D(3) in the kidney, which then proceeds to bone and intestine where it mobilizes calcium. Thus 1,25-(OH)(2)D(3) is a hormone drived from vitamin D which is responsible for the mobilization of calcium and the parathyroid hormone serves as its tropic hormone. Radioactive dihydrotachysterol(3) has been synthesized and shown to be hydroxylated by the liver to its 25-hydroxy derivative. This metabolite appears to be the metabolically active form of this analog of vitamin D. It is effective in animals which are nephrectomized and hence does not require 1-hydroxylation. Its effectiveness as a substitute for the 1,25-(OH)(2)D(3) results from the factthat its A ring is rotated 180, placing its 3beta-OH in the position occupied by the 1alpha-OH of 1,25-(OH)(2)D(3).

Impacts
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Publications

Progress 01/01/71 to 12/30/71

Outputs
A metabolite of 25-hydroxycholecalciferol (25-HCC) has been isolated from the intestines of chicks and unequivocally identified as 1,25- dihydroxycholecalciferol (1,25-DHCC). This metabolite has been demonstrated to be the metabolically active form of vitamin D in the stimulation of intestinal calcium absorption and in the stimulation of calcium mobilization from bone. Its action on intestine is not inhibited by actinomycin D and therefore its effect in this organ is not dependent upon RNA and protein synthesis. The conversion of 25-HCC to the 1,25-DHCC compound takes place in kidney mitochondria and is blocked by the prior administration of actinomycin D and cycloheximide. The enzyme system responsible for the conversion is carbon monoxide sensitive and requires reduced pyridine nucleotide. The hydroxylation is regulated by dietary calcium and consequently plasma calcium concentration. Diets low in calcium bring about the hydroxylation of 25-HCC to 1,25-DHCC. Diets high in calcium, however, bring about the hydroxylation of 25-HCC to a newmetabolite, as yet unidentified. Strontium feeding inhibits intestinal absorption of calcium primarily by inhibiting the conversion of 25-HCC to 1,25-DHCC. This inhibition is overcome by the administration of 1,25-DHCC to strontium-fed animals.

Impacts
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Publications

Progress 01/01/70 to 12/30/70

Outputs
Two new metabolites of vitamin D have been isolated and identified as 21,25-dihydroxycholecalciferol and 25,26-dehydroxycholecalciferol. Another new metabolite has been described, which is the probable metabolically active form of vitamin D in the intestine. It has been highly purified and its biological activity tested. A new chromatographic method for vitamin D and its metaboliteshas been devised, using gel liquid partition chromatography. Using this system,it was shown that the peak 5 or polar metabolite of vitamin D found in the intestine is (1) made in the kidney, (2) its synthesis is blocked by actinomycin-D administration, (3) it functions in the intestine in the presence of actinomycin and thus represents the metabolically active form. Retinol is converted to retinoic acid in the kidney before retinoic acid appears in the plasma, suggesting it to be a candidate for the active form of vitamin A in growth.

Impacts
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Publications

Progress 01/01/69 to 12/30/69

Outputs
The circulating active form of vitamin D(2) has been isolated and identified as 25-hydroxyergocalciferol. It is extremely antirachitic (60 i.u./micrograms) andfunctions more rapidly than the parent vitamin. The 25-hydroxycholecalciferol has been synthesized in quantity and has been shown to be effective in the cure of vitamin D resistant hypoparathyroidism. It has also been shown to prevent milk fever disease in dairy cattle, thus eliminating one of the most troublesomeveterinarian problems in the dairy industry. The site of vitamin D action on the intestine has been clearly delineated and an enzyme system in the microvilliof intestine which appears after vitamin D has been discovered. The role of vitamin D on intestinal nuclear activity has been shown to be related to an increased chromatin template activity for the synthesis of RNA. At least three new metabolites of vitamin D have been discovered in intestine and bone but their exact role has not been determined.

Impacts
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Publications