Sally A. Holick

1 alpha,25-dihydroxyvitamin D3 induces maturation of the human monocyte cell line U937, and, in association with a factor from human T lymphocytes, augments production of the monokine, mononuclear cell factor.

The monocyte factor, interleukin 1, or other factors homologous with interleukin 1, modulates functions of a variety of cells, including T and B lymphocytes, synovial cells, and chondrocytes. We have reported that a human monocyte cell line, U937, produces interleukin 1 when incubated with a soluble factor from lectin-stimulated T lymphocytes. We have also shown that U937 cells have a specific cytosolic receptor for 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25[OH]2D3). We now report that 1 alpha,25(OH)2D3(10(-11)-10(-10) M) induces maturational changes in the U937 cells similar to those produced by conditioned medium from lectin-stimulated T lymphocytes (increase in Fc receptors and OKM1 binding and decrease in proliferation), but does not induce monokine production as measured by mononuclear cell factor activity. 1 alpha,25(OH)2D3 is 200-300-fold more effective than 25-hydroxyvitamin D3, which is consistent with the known biological potency of these vitamin D3 metabolites. 1 alpha,25(OH)2D3 and the lymphokine together markedly augment maturational effects and, in addition, augment monokine production. The specificity of the interaction is further demonstrated by the lack of augmentation of monokine production with 1 beta,25-dihydroxyvitamin D3 in the presence of lymphokine. These interactions of a classical hormone and the hormonelike product(s) of the immune system with U937 cells serve as a model for human monocyte/macrophage differentiation and suggest a role for these interactions in some aspects of inflammation.

Synthesis of (6-3H)-1alpha-hydroxyvitamin D3 and its metabolism in vivo to (3H)-1alpha,25-dihydroxyvitamin D3

[6-3H]-1alpha-Hydroxyvitamin D3 was chemically synthesized and its full biological activity and radiochemical purity were demonstrated. With the use of this preparation it has been possible to demonstrate in vivo that in rats the [6-3H]-1Alpha-hydroxyvitamin D3 is converted to [6-3H]-1alpha,25-dihydroxyvitamin D3, the natural hormone. In fact, in the intestine and bone of rats given 32 picomoles of [6-3H]-1alpha-hydroxyvitamin D3 each day for 6 days, more than 80 percent of the lipid-soluble radioactivity exists as [6-3H]-1alpha,25-dihydroxyvitamin D3, a finding that suggests that much of the biological effectiveness of 1alpha-hydroxyvitamin D3 is due to its conversion to 1alpha,25-dihydroxyvitamin D3.

The photoproduction of 1 alpha ,25-dihydroxyvitamin D3 in skin. An approach to the therapy of vitamin-D-resistant syndromes.

Cutaneous 7-dehydrocholesterol, exposed to ultraviolet radiation, converts to previtamin D3, which in turn converts in skin to vitamin D3 and is carried into the circulation. We investigated the feasibility of the photochemical conversion in skin of hydroxylated derivatives of 7-dehydrocholesterol - such as 1 alpha, 25-dihydroxy-7-dehydrocholesterol (1 alpha, 25-(OH)2-7-DHC) - to the corresponding hydroxylated previtamin as an alternative method of delivery of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2-D3) to subjects who are deficient in the endogenous metabolite. In human volunteers and in vitamin-D-deficient rats [24-3H] 1 alpha, 25-(OH)2-D3 appeared in blood after [24-3H] 1 alpha, 25-(OH)2-7-DHC was applied to the skin and exposed to ultraviolet radiation. In anephric rats, intestinal calcium absorption and serum calcium levels were elevated after a topical dose of 1 alpha, 25-(OH)2-7-DHC combined with ultraviolet phototherapy. Delivery of equivalent doses of 1 alpha, 25-(OH)2-D3 through the skin and orally showed that there was more prolonged stimulation in intestinal calcium absorption and serum calcium elevation after cutaneous administration. The photochemical conversion of precursors may be useful in the treatment of patients with impaired vitamin-D metabolism.

Evaluation of a photolabile derivative of 1,25-dihydroxyvitamin D3 as a photoaffinity probe for 1,25-dihydroxyvitamin-D3 receptor in chick intestinal cytosol.

We evaluated the viability of 1 alpha, 25-dihydroxyvitamin D3-3 beta-[N-(4-azido-2-nitrophenyl)glycinate] (1,25-(OH)2-D3-ANG), an analog of 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2-D3) as a photoaffinity probe for 1,25-(OH)2-D3 receptor in chick intestinal cytosol. A competitive-binding assay revealed that chick intestinal cytosolic 1,25-(OH)2- D3 receptor bound to 1,25-(OH)2-D3-ANG approximately 20-times less effectively than it did to 1,25-(OH)2-D3. Irradiation of 1,25-(OH)2-D3- ANG in the presence of chick intestinal cytosolic preparation significantly diminished subsequent binding to 3H-1,25-(OH)2-D3, suggesting that the photoaffinity analog was covalently attached to the receptor. Therefore the nitroarylazide derivative of 1,25-(OH)2-D3 may be a valuable photoaffinity probe for the characterization of the 1,25-(OH)2-D3 receptor.

Synthesis of a photoaffinity-labelled analogue of 1,25-dihydroxyvitamin D3

The synthesis of a biologically active analogue of 1,25-dihydroxyvitamin D3 containing a photolabile azidonitrophenyl group is described.

A simple method for generation of antibodies with specificity for 1,25-dihydroxyergocalciferol and 1,25-dihydroxycholecalciferol

A simple method for production of antisera with high affinity and selectivity for 1 alpha, 25-dihydroxyergocalciferol and 1 alpha, 25-dihydroxychole-calciferol is described. 1 alpha-Hydroxy-25,26,27-trisnorcholecalciferol-24-oic acid was coupled directly to bovine serum albumin. Rabbits immunized with this conjugate rapidly produced antibodies that bound 3H-1 alpha,-25-dihydroxycholecalciferol with high affinity and demonstrated nearly equal reactivity with 1 alpha, 25-dihydroxyergocalciferol and poor reactivity with 25-hydroxycholecalciferol; 24,25-dihydroxycholecalciferol; 25,26-dihydroxycholecalciferol; and 1 beta,25-dihydroxycholecalciferol. The use of one of these antisera has led to the development of a specific assay for 1 alpha,25-dihydroxyergocalciferol and 1 alpha,25-dihydroxycholecalciferol in human serum.

Synthesis of 19-acetylthio-3-β-acetoxy-9,10-secochola-1(10),5Z,7E-triene, and 1-β-acetylthio-3-β-acetoxy-9,10-secochola-5Z,7E,10(19)-triene: an approach to 1β- and 19-thiolation of the vitamin D skeleton

Reaction of 1-α-methylsulphonyloxy or 1-α-(p-tolylsulphonyloxy) derivatives of vitamin D3-acetate with KSAc in dimethyl sulphoxide resulted exclusively in 19-acetylthiolation; the same reaction with 1-α-p-tolylsulphonyloxy-(6R)-methoxycyclovitamin D3 and its corresponding 25-hydroxy derivatives offered a selective route to 1-β-thiol analogues of vitamin D3 and 25-hydroxyvitamin D3, both of which were capable of competing with 1α,25-dihydroxyvitamin D3 for chick intestinal cytosolic receptor.