Biauw Keng Tan

The effect of vitamin D analogs and of vitamin D-binding protein on lymphocyte proliferation

In the absence of vitamin D-binding protein (DBP), 1,25-(OH)2D3 at 10(-12) M significantly inhibited the [3H]thymidine incorporation in human lymphocytes during mixed lymphocyte cultures (MLC) or after phyto-hemaglutinin (PHA) stimulation. In the presence of a physiological concentration of DBP (5 x 10(-6) M), the concentration of 1,25-(OH)2D3 required for inhibition was 10(-10) M (for PHA-cultures) and 10(-9) M (for MLC). Several vitamin D analogs were compared for their inhibitory action on PHA stimulation. In the absence of DBP, the concentration necessary for 50% inhibition of [3H]thymidine incorporation ranged from 10(-12) M [1,25-(OH)2D3 and 24,24-F2-1,25-(OH)2D3], over 10(-10) M [1,24R, 25-(OH)3D3; 1,25S, 26-(OH)3D3 and 26,27-F6-1,25-(OH)2D3] and 10(-8) M [25 OHD3 and 24,25-(OH)2D3] to 10(-6) M [calcitriol-lactone]. This rank order correlates with the binding affinity of the various analogs to the cytoplasmic 1,25-(OH)2D3-receptor. DBP counteracted the inhibitory effect of all analogs and the degree of counteraction was directly proportional to the binding affinity between DBP and the vitamin D analog. DBP thus decreased the in vitro inhibitory action of 1,25-(OH)2D3 and its analogs on lymphocyte proliferation. Of all analogs tested, only 1,25-(OH)2D3 had a significant effect at a physiological concentration.

Differentiation induction of human leukemia cells (HL60) by a combination of 1,25-dihydroxyvitamin D3 and retinoic acid (all trans or 9-cis)

1,25(OH)2D3 and two stereoisomers of retinoic acid, all trans and 9-cis retinoic acid, are regulators of cell proliferation and differentiation. The aim of this study was to evaluate the effects of a combination of 1,25(OH)2D3 and retinoic acid (all trans or 9-cis) on proliferation and cell differentiation of the human promyelocytic leukemia cell line HL60, and to test the reversibility of the induced differentiation. Cell proliferation was inhibited as expected by 1,25(OH)2D3 and all trans retinoic acid alone (IC50 of cell survival was 4 x 10(-7) M, 9 x 10(-6) M and 9 x 10(-7) M for 1,25(OH)2D3, all trans and 9-cis retinoic acid, respectively). Combination of 1,25(OH)2D3 and either form of retinoic acid resulted in a partially additive decrease in cell proliferation. 1,25(OH)2D3 induced a monocytic differentiation (100% CD14+ cells with 10(-7) M 1,25(OH)2D3), while retinoic acid led to a predominantly granulocytic differentiation (36 and 42% CD67+ cells with 10(-6) M all trans and 9-cis retinoic acid, respectively). Additive effects on differentiation were observed upon combination of subtherapeutical doses of the drugs, achieving a mainly monocytic population, demonstrating the dominant role of 1,25(OH)2D3 in determining the direction of differentiation. The effects on proliferation and differentiation of the solitary drugs were reversible, while the proliferation arrest and differentiation induced by the combination persisted and even progressed after withdrawal of the drugs. We conclude that 1,25(OH)2D3 and retinoic acid (all trans or 9-cis) exert additive effects on inhibition of proliferation and induction of cell differentiation of HL60 cells, leading to a persistent differentiation, even after drug withdrawal.

Nrp2 deficiency leads to trabecular bone loss and is accompanied by enhanced osteoclast and reduced osteoblast numbers

Neuropilin 1 (Nrp1) and Nrp2 are transmembrane receptors that can bind class 3 semaphorins (Sema3A-G) in addition to VEGF family members to play important roles in axonal guidance, vascularization and angiogenesis, as well as immune responses. Moreover, recent evidence implicates Sema3A/Nrp-mediated signaling in bone regulation. However, to date the expression of Nrp2 in bone has not been investigated and a possible role for Nrp2 in the maintenance of bone homeostasis in vivo remains unexplored. Here we show that Nrp2, together with its possible coreceptors (Plexin A family members and Plexin D1) and class 3 semaphorin ligands, were expressed during in vitro osteogenic differentiation of bone marrow stromal cells. Moreover, Nrp2 transcript and protein levels were highly induced in hematopoietic bone marrow cell-derived osteoclast cultures. Osteoblastic as well as osteoclastic Nrp2 expression was confirmed by immunohistochemistry of the long bones of mice. Interestingly, Nrp2 knockout mice were characterized by a low bone mass phenotype which was accompanied by an increased number of osteoclasts and a decreased osteoblast count. Collectively, these data point to a physiological role for Nrp2 in bone homeostasis.

Nonhypercalcemic vitamin D analogs: interactions with the vitamin D-binding protein.

The natural vitamin D hormone, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3), not only regulates serum and bone calcium homeostasis but is probably also a paracrine factor in several cells and tissues including skin, immune system, placenta and brain, where it stimulates cell differentiation and inhibits cell proliferation. Several structural analogs of 1 alpha, 25-(OH)2D3 not only have superagonist activity but also display a selective action profile: indeed they maintain or have increased activity on cell differentiation/proliferation but also have substantially decreased calcemic activity when compared to 1 alpha,25-(OH)2D3. This decreased calcemic activity is partially due to mere pharmacological reasons: because of low binding affinity for the plasma vitamin D-binding protein, a more rapid extracellular metabolism and increased cellular uptake is possible when compared to 1 alpha,25-(OH)2D3. Their short extracellular half-life combined with comparable or enhanced transactivation potency together with analog-and cell-type-specific intracellular metabolism can probably explain why some analogs have a unique combination of superagonist activity and specific action profile with favorable dissociation of differentiation versus calcemic potency.

The biological activity of 23-oxa-, 23-oxa-24-oxo-, and 23-thia-dihydroxyvitamin D3

Three analogs of 1 alpha,25-(OH)2D3 with an oxygen or another heteroatom at position 23 were synthesized in search of separating the cell-differentiating from the calcemic effects of the vitamin D hormone. Their ability to induce superoxide production in human myeloid leukemia cells (HL-60) was 1 alpha,25-(OH)2D3 > 23-oxa-24-oxo-1 alpha,25-(OH)2D3 > 23-thia-1 alpha,25-(OH)2D3 > 23-oxa-1 alpha, 25-(OH)2D3. 23-oxa-24-oxo-1 alpha, 25(OH)2D3 was slightly more potent than 1 alpha,25-(OH)2D3 in inhibiting cell proliferation in MCF-7 cells and 23-thia- and 23-oxa-1 alpha,25(OH)2D3 were less potent. Their in vitro potency to produce osteocalcin in MG-63 cells was 1 alpha,25-(OH)2D3 > 23-oxa-24-oxo-1 alpha,25-(OH)2D3 > 23-thia-1 alpha,25-(OH)2D3 = 23-oxa-1 alpha,25-(OH)2D3. All three analogs had reduced receptor and DBP affinity compared to 1 alpha,25-(OH)2D3. When these analogs were injected in rachitic chicks, only little calcemic effects were observed. The introduction of a heteroatom in carbon 23 of 1 alpha,25-(OH)2D3 thus creates analogs with dissociated action on cell differentiation and calcium homeostasis.