Analysis of Vitamin D3 Analogues for Effects on Differentiation and Vitamin D Receptor Expression.

Brian A. McCarthy

Introduction:

Vitamin D is a nonpolar, secosteroid derivative of cholesterol activated through 2 stages of metabolism before reaching actual hormonal form. It is the vitamin that mediates intestinal calcium absorption, bone calcium metabolism, and probably muscle activity. It usually acts as a hormone precursor and is isolated from fish liver oils and used in the treatment and prevention of rickets.[1]

1,25-OH2D3 is not truly a vitamin because it is not required in the diet. In skin cells 7-dehydrocholesterol, an intermediate in cholesterol biosynthesis, undergoes ultraviolet photo lysis into D3. Activation involves an intramolecular oxidoreductase and the opening of its second ring. First there is the loss of a hydrogen to 7-dehydrocholesterol, provitamin D3, and finally the ring opens into activated 7-dehydrocholesterol also called 1,25-dihydroxyvitamin D3 (1,25-OH2D3),(D3) or cholecalciferol. [2] This mechanism has been known for decades; the second ring is opened between C9 and C10, there is a conversion of a methyl group at C10 to a methylene (=CH2) and a hydrogenation of C9. The alcohol group at C3 must be free or is esterified, capable of being hydrolyzed by the organism. Only two forms are important for human nutrition; vitamin D2, calciferol; activated ergosterol and Vitamin D3.[3]

D3 has genomic effects (hormone/promoter via cognate nuclear receptor; VDR) and in some cell lines, non genomic (plasma membrane Ca++). 1,25-OH2D3 migrates to target cells in the intestine and osteoblasts. In the intestine transcription is stimulated and a protein that stimulates calcium absorption in the blood is synthesized and in osteoblasts calcium uptake is stimulated for deposition as calcium phosphate.[2] Proteins, usually found in the cytoplasm, that specifically bind calcitriol, migrate to the nucleus, and regulate transcription of specific segments of DNA. Vitamin D is converted in the liver and kidney to calcitriol and ultimately acts through these receptors.[1]

Vitamin D was first recognized for its ability to cure rickets in 1862 when Trousseau recommended cod liver oil as a remedy and is one of the first reported cures by medication. [4] In one of the earliest epidemiologic studies, Palm associated sunlight and rickets thru geographic studies [5]. However not until x-ray studies established the light of a mercury vapor lamp could cure extreme rickets was the importance of sunlight appreciated.[6]

1,25-OH2D3 utilizes its own cognate nuclear receptor to stimulate changes in transcription that alter cell behavior. Vitamin D is unique among the steroids in that the protein product of the VDR is a calcium binding protein. Excess vitamin D can lead to hypercalcemia and calcification of soft tissues. The amount of VDR present is mainly dependent upon the stability of the receptor, rather than the quantity produced.

Complementary DNA clones encoding the human vitamin D receptor were reported in 1988 after isolation from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3' noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of approximately equal to 4600 bp. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor. [7]

Mutations in the VDR can cause pathology. In one case of hereditary vitamin D-resistant rickets (HVDRR) northern blot analysis showed that a normal size VDR transcript was expressed; however, Tridiated [H3] 1,25-OH2D3-binding levels were very low and Western blot analysis failed to detect any VDR protein. Subsequently a single base substitution was discovered through DNA analysis. A unique C to T base change corresponding to nucleotide 218 of the VDR cDNA changed the codon for arginine (CGA) to a stop codon (TG